A-21270

antibody from Invitrogen Antibodies

Targeting: GOLGA1 golgin-97, MGC33154

Provider product page for A-21270

Western blot

Immunocytochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [183]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [18]
Other assay [51]

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Product number: A-21270 - Provider product page
Provider: Invitrogen Antibodies
Product name: Anti-Golgin-97 Monoclonal Antibody (CDF4)
Antibody type: Monoclonal
Antigen: Other
Description: Anti-human golgin-97 antibodies were originally isolated from the serum of a patient with the autoimmune disease known as Sjögren's syndrome. These antibodies recognize a 97 kDa protein called golgin-97, a member of the granin family of proteins and a peripheral membrane protein localized on the cytoplasmic face of the Golgi apparatus. Because the antibody recognizes a protein unique to the Golgi apparatus of most vertebrate species, it is useful for immunodetection and identification of the Golgi apparatus in cells. To prepare a stock solution, reconstitute the antibody in 0.5-1 mL of phosphate-buffered saline, pH 7.4, containing 1% BSA. Store the solution for up to two weeks at 4°C with the addition of 2 mM sodium azide. For longer storage, divide the solution into single-use aliquots and freeze at -20°C.
Reactivity: Human, Mouse, Rat
Host: Mouse
Isotype: IgG
Antibody clone number: CDF4
Vial size: 100 µg
Storage: -20°C

GOLGA1 protein structure - A-21270 shown in red.

Supportive validation

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Experimental details: Knockdown of Golgin-97 was achieved by transfecting T-47D with Golgin-97 specific siRNAs (Silencer® select Product # S5940, S5939). Western blot analysis (Fig. a) was performed using Nuclear enriched extracts from the Golgin-97 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with Golgin-97 Monoclonal Antibody (CDF4) (Product # A21270, 1:1000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Golgin-97.

Supportive validation

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Experimental details: Western blot was performed using Anti-Golgin-97 Monoclonal Antibody (CDF4) (Product # A21270) and a 97kDa band corresponding to Golgin-97 was observed along with uncharacterized bands (*) at 55 kDa across cell lines and tissues tested. Whole cell extracts (30 µg lysate) of T-47D (Lane 1), HeLa (Lane 2), MCF7 (Lane 3), COLO 205 (Lane 4), SW480 (Lane 5), Mouse Pancreas (Lane 6), Mouse Testis (Lane 7) and Rat Testis (Lane 8) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177,1:20000 dilution) using the iBright FL 1000 (Product # A32752). Relative expression of Golgin-97 was observed to be high in Mouse Testis in comparison to no expression in Mouse Pancreas. Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580).

Supportive validation

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Experimental details: HeLa cells were plated on coverslips overnight. The next day cells were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit (Product # R37602) according to protocol. Cells were then incubated with 1 µg/mL anti-golgin-97 antibody (Product # A-21270) for labeling of Golgi for 30 min at room temperature and washed three times with dPBS. Cells were then incubated with a 1:1000 dilution of FITC-labeled secondary antibody (Product # A16079) for 30 min and washed three times in dPBS. Cells were labeled with NucBlue® Live (Product # R37605) and ActinRed™ 555 ReadyProbes® reagent (Product # R37112) according to protocol. Coverslips were then mounted using ProLong® Gold antifade reagent (Product # P36930). Images were taken using the EVOS® FL Auto Imaging System and a 40X Coverslip corrected objective (Product # AMEP4699).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: HeLa cells were plated on coverslips overnight. The next day cells were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit (Product # R37602) according to protocol. Cells were then incubated with 1 µg/mL anti-golgin-97 antibody (Product # A-21270) for labeling of Golgi for 30 min at room temperature and washed three times with dPBS. Cells were then incubated with a 1:1000 dilution of FITC-labeled secondary antibody (Product # A16079) for 30 min and washed three times in dPBS. Cells were labeled with NucBlue® Live (Product # R37605) and ActinRed™ 555 ReadyProbes® reagent (Product # R37112) according to protocol. Coverslips were then mounted using ProLong® Gold antifade reagent (Product # P36930). Images were taken using the EVOS® FL Auto Imaging System and a 40X Coverslip corrected objective (Product # AMEP4699).

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Experimental details: BPAE cells were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit (Product # R37602). The mitochondrial ox-phos complex V inhibitor protein was stained using primary antibody: Mouse anti ATP Synthase Subunit IF1 Monoclonal Antibody, (clone 5E2D7) (Product # A-21355) detected with a goat anti-mouse Alexa Fluor® 488 secondary antibody (Product # A-11029). Actin was stained using Alexa Fluor® 594 phalloidin (Product # A12381) and nuclei were stained using NucBlue Live Cell Stain (Product # R37605). Slides were mounted using ProLong® Gold Antifade Kit (P7481) and images were acquired on the FLoid™ Cell Imaging Station (Product # 4471136).

Supportive validation

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Experimental details: Human dermal fibroblasts, neonatal (HDFn) (C-004-5C) were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit (Product # R37602). Golgi staining was done using an anti-Golgin-97 primary antibody (Product # A-21270) and a goat anti-mouse Alexa Fluor® 488 secondary antibody (Product # A-11029). Actin was stained using Alexa Fluor® 594 phalloidin (Product # A12381) and nuclei were stained using NucBlue™ Live Cell Stain (Product # R37605). Slides were mounted using ProLong® Gold antifade kit (P7481) and images were acquired on the FLoid™ Cell Imaging Station (Product # 4471136).

Supportive validation

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Experimental details: HDFn cells were fixed, permeabilized, and blocked using the Image-iT® Fixation/Permeabilization Kit (Product # R37602). Golgi were labeled with an anti-golgin 97 antibody (Product # A-21270) followed by detection with an Alexa Fluor® 488 secondary (Product # A-11029). Cells were also stained with Alexa Fluor® 594 phalloidin (Product # A12381) to label actin and NucBlue Fixed (Product # R37606) to label nuclei. Finally, cells were mounted in ProLong® Gold antifade reagent.

Supportive validation

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Experimental details: HDFn cells were fixed, permeabilized, and blocked using the Image-iT® Fixation/Permeabilization Kit (Product # R37602). Golgi were labeled with an anti-golgin 97 antibody (Product # A-21270) followed by detection with an Alexa Fluor® 488 secondary (Product # A-11029). Cells were also stained with Alexa Fluor® 594 phalloidin (Product # A12381) to label actin and NucBlue Fixed (Product # R37606) to label nuclei. Finally, cells were mounted in ProLong® Gold antifade reagent.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: HeLa cells were plated on coverslips overnight. The next day cells were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit (Product # R37602) according to protocol. Cells were then incubated with 1 µg/mL anti-golgin-97 antibody (Product # A-21270) for labeling of Golgi for 30 min at room temperature and washed three times with dPBS. Cells were then incubated with a 1:1000 dilution of FITC-labeled secondary antibody (Product # A16079) for 30 min and washed three times in dPBS. Cells were labeled with NucBlue® Live (Product # R37605) and ActinRed™ 555 ReadyProbes® reagent (Product # R37112) according to protocol. Coverslips were then mounted using ProLong® Gold antifade reagent (Product # P36930). Images were taken using the EVOS® FL Auto Imaging System and a 40X Coverslip corrected objective (Product # AMEP4699).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: HeLa cells were plated on coverslips overnight. The next day cells were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit (Product # R37602) according to protocol. Cells were then incubated with 1 µg/mL anti-golgin-97 antibody (Product # A-21270) for labeling of Golgi for 30 min at room temperature and washed three times with dPBS. Cells were then incubated with a 1:1000 dilution of FITC-labeled secondary antibody (Product # A16079) for 30 min and washed three times in dPBS. Cells were labeled with NucBlue® Live (Product # R37605) and ActinRed™ 555 ReadyProbes® reagent (Product # R37112) according to protocol. Coverslips were then mounted using ProLong® Gold antifade reagent (Product # P36930). Images were taken using the EVOS® FL Auto Imaging System and a 40X Coverslip corrected objective (Product # AMEP4699).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: HeLa cells were plated on coverslips overnight. The next day cells were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit (Product # R37602) according to protocol. Cells were then incubated with 1 µg/mL anti-golgin-97 antibody (Product # A-21270) for labeling of Golgi for 30 min at room temperature and washed three times with dPBS. Cells were then incubated with a 1:1000 dilution of FITC-labeled secondary antibody (Product # A16079) for 30 min and washed three times in dPBS. Cells were labeled with NucBlue® Live (Product # R37605) and ActinRed™ 555 ReadyProbes® reagent (Product # R37112) according to protocol. Coverslips were then mounted using ProLong® Gold antifade reagent (Product # P36930). Images were taken using the EVOS® FL Auto Imaging System and a 40X Coverslip corrected objective (Product # AMEP4699).

Supportive validation

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Experimental details: HeLa cells. Anti-human Golgin-97, mouse monoclonal CDF4, Alexa Fluor® 430 goat anti-mouse IgG, Alexa Fluor® 594 phalloidin, and SYTOX® Green nucleic acid stain. Fixed, permeabilized and RNase-treated HeLa cells were labeled with mouse monoclonal anti-human Golgin-97 monoclonal antibody (Product # A-21270) and visualized with the yellow-fluorescent Alexa Fluor® 430 goat anti-mouse IgG antibody (Product # A-11063). The F-actin was labeled with red-fluorescent Alexa Fluor® 594 phalloidin (Product # A12381) and the nuclei were stained with the SYTOX® Green nucleic acid stain (Product # S7020). This multiple-exposure image was acquired using a Nikon Diaphot fluorescent microscope equipped with a Quantix CCD camera.

Supportive validation

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Experimental details: HeLa cells labeled with SYTOX® Orange nucleic acid stain, anti-Golgin-97 antibody and detected using Alexa Fluor® 647 goat anti-mouse IgG antibody. Fixed, permeabilized HeLa cells were labeled with anti-Golgin-97 polyclonal antibody (Product # A-21270) and detected using Alexa Fluor® 647 goat anti-mouse IgG antibody (Product # A-21235). The cells were counterstained with SYTOX® Orange nucleic acid stain (Product # S11368).

Supportive validation

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Experimental details: HeLa cells labeled with anti-golgin-97 antibody and detected using Alexa Fluor® 488 Goat Anti-Mouse IgG antibody. Fixed, permeabilized HeLa cells were labeled with anti-golgin-97 monoclonal antibody (Product # A-21270) and detected using Alexa Fluor® 488 Goat Anti-Mouse IgG antibody (Product # A-11001). The cells were counterstained with DAPI (D1306, D3571, D21490). (Product # A-21270).

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Experimental details: Muntjac skin cell labeled in three different colors. Golgi bodies in a muntjac skin cell were labeled with anti-Golgin-97 monoclonal antibody (Product # A-21270) and visualized with green-fluorescent Alexa Fluor® 488 Goat Anti-Mouse IgG1 antibody (Product # A-21121). Filamentous actin was labeled with Alexa Fluor® 680 phalloidin (Product # A22286, pseudocolored purple). Nuclei were stained with blue-fluorescent DAPI (Product # D1306, D3571, D21490). (Product # A-21270).

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Experimental details: Increased label specificity and resolution afforded by Image-iT® FX signal enhancer. Fixed and permeabilized MRC-5 human lung fibroblast cells were labeled with mouse anti-human Golgin-97 primary antibody (Product # A-21270), then visualized with either FITC-conjugated goat anti-mouse IgG (F2761, top row) or Alexa Fluor® 488 Goat Anti-Mouse IgG (Product # A-11001, bottom row). Cells on the right were treated with Image-iT® FX signal enhancer (Product # I36933) prior to primary antibody incubation (Product # A-21270).

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Experimental details: Golgi and actin staining in HeLa cells. Golgi in fixed and permeabilized HeLa cells labeled with anti-golgin-97 monoclonal antibody (Product # A-21270) and visualized with green-fluorescent Alexa Fluor® 488 Goat-Anti-Mouse IgG (Product # A-11001). Actin was stained with red-fluorescent Alexa Fluor® 594 phalloidin (Product # A12381); nuclei were stained with blue-fluorescent DAPI (Product # D1306, D3571, D21490). Treatment with Image-iT® FX signal enhancer (Product # I36933) largely eliminates nonspecific dye binding (bottom) as compared to untreated slide (top).

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Experimental details: Immunofluorescence analysis of Golgin-97 was performed using 70% confluent log phase T-47D cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Golgin-97 Monoclonal Antibody (CDF4) (Product # A21270) at 1:100 dilution in 0.1% BSA, incubated at 4 °C overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing golgi localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification in EVOS™ M7000 Imaging System (Product # AMF7000) and externally deconvoluted (D.Sage et al. / Methods 115 (2017) 28-41).

Supportive validation

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Main image
Experimental details: Human dermal fibroblasts, neonatal (HDFn) (C-004-5C) were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit (Product # R37602). Golgi staining was done using an anti-Golgin-97 primary antibody (Product # A-21270) and a goat anti-mouse Alexa Fluor® 488 secondary antibody (Product # A-11029). Actin was stained using Alexa Fluor® 594 phalloidin (Product # A12381) and nuclei were stained using NucBlue Live Cell Stain (Product # R37605). Slides were mounted using ProLong® Gold antifade kit (P7481) and images were acquired on the FLoid Cell Imaging Station (Product # 4471136).

Supportive validation

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Experimental details: Golgi in HeLa cells detected with Alexa Fluor® 546 tyramide. Golgi in HeLa cells detected with Alexa Fluor® 546 tyramide. Cells were fixed and permeabilized, then labeled with anti-human Golgin-97 polyclonal antibody (Product # A-21270) and detected using HRP-conjugated goat anti-mouse IgG antibody and Alexa Fluor® 546 tyramide, which are components of the TSA Kit #3 (Product # T-20913). The nuclei were counterstained using DAPI (Product # D1306, D3571, D21490). The images were acquired using filters appropriate for DAPI and Alexa Fluor® 546 and processed using MetaMorph software from Universal Imaging Corp.

Supportive validation

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Experimental details: Figure 3 Golgi fragmentation in apoptotic HT29 cells. Cells were treated with serial concentrations of geoditin A for 24 h, fixed in 3% paraformaldehyde (PFA) and incubated with antibody against Golgi marker protein, golgin-97 and visualized by secondary antibody IgG conjugated to Alexa-647. Nuclei were counterstained with DAPI. Typical Golgi stack is shown at the juxtanuclear region of the untreated HT29 cells (a), while Golgi fragmentation becomes prominent in geoditin A-treated in a dose-dependent manner (b, c, and d for 10, 20 and 40 mug/mL, respectively).

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Supportive validation

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Experimental details: Figure 5 Retention of cadherin 23 by PIST in trans-golgi network in transfected COS-7 cells . A. Myc-tagged cadherin 23 localizes in the cytoplasm. B. In the presence of EGFP-tagged PIST, Myc-tagged cadherin 23 co-localizes with EGFP-PIST and a trans-golgi network marker golgin-97. Myc-cadherin 23 was stained with rabbit anti-cadherin 23 antibody and golgin-97 was stained with mouse anti-golgin-97 antibody. Scale bar, 20 mum.

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Experimental details: Figure 7 Intracellular localization of PLD4 protein exogenously expressed in cultured cell lines. A, EGFP fluorescent imaging (green) and DAPI nuclear staining (blue) of HEK293 cells transfected with pEGFP-PLD4. PLD4 is localized in a meshwork-like structure and around the periphery of nuclei. B and C, immunocytochemical localization of PLD4 protein (green) and either calnexin, an ER marker (red) (B) or golgin 97, a trans -Golgi marker (red) (C), in HeLa cells transfected with pcDNA3-PLD4. PLD4 is colocalized with calnexin in the ER and with golgin 97 in the Golgi complex. Scale bars = 10 um.

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Experimental details: Figure 1 Podocin localizes to endosomal vesicles. Immunofluorescence for transiently expressed, V5- (a-c, e) or Flag-tagged (d) podocin using anti-V5 or anti-Flag antibody in HeLa cells. Costainings of endogenous calnexin (a), endogenous golgin-97 (b), endogenous EEA1 (c), Lysotracker Red (d) and eGfp-tagged and transiently expressed CD63 (e) as markers for the endoplasmatic reticulum, Golgi apparatus, early endosomes, acidic organelles and late endosomes respectively, displayed that podocin localizes to the endosomal compartment. Analogously to HeLa cells, transiently expressed V5-tagged podocin colocalizes with eGFP tagged CD63/LAMP3 in cultured human podocytes (f).

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Experimental details: Figure 8 Endogenous localization of SelS includes a perinuclear accumulation. A , U251 cells or HepG2 cells were examined for the localization of endogenous SelS protein by immunofluorescence as described in Materials and Methods . Images were taken at 40x magnification. B , Costaining of SelS (green) and the Golgi (red) was performed using alpha-SelS and alpha-golgin p97 antibodies in HepG2 cells. Regions of colocalization are indicated in yellow (40x magnification).

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Experimental details: Fig. 6 Characterization of exs derived from ascites using antibodies. (a) Presence of characteristic exs markers Alix, Hsp70 and Tsg101 as well as CD63, and absence of ""antimarker"" Golgin A1 (present in the cells of origin but absent from exs) was verified by western blotting (WB) in cells (P 0.2 fraction) and exs from ovarian cancer patient #2. (b) TEM of exs isolated from ascites of ovarian cancer patient #2. Exs marker CD63 is detected at the surface of exs by secondary antibodies conjugated to 10-nm gold particles. (c) CD63 + , exs derived from ascites of ovarian cancer patient #2 can be detected by FC analysis of CD63-PE antibody-stained exs (right panel). Left panel, exs stained with isotype control antibody-PE. (d) Double labeling of exs from ascites of patient #1 by CFSE and EpCAM-APC antibody reveals feasibility of double labeling and demonstrates that only 11.5% of CFSE + exs are EpCAM + . (e) Summary of timing of the presented protocol. Each step is marked in the time scale by different color. For collection of EVs from conditioned media, cells are plated a day ahead. Isolation of exs can be achieved in 1 day. Setting up the instrument and FC data acquisition can be completed within the morning of the second day. - F, 0.22 um filtered; - NF, 0.22 um non-filtered.

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Experimental details: Fig. 2 Beclin 1 localizes to endosomes in neurons. Representative images showing colocalization of beclin 1 with ( a ) Rab5-GFP, colocalization coefficient 0.49 +- 0.03, ( b ) Rab7-GFP, colocalization coefficient 0.50 +- 0.03 or ( c ) Golgin 97, colocalization coefficient 0.13 +- 0.01 in primary hippocampal neurons. Regions in the red boxes are enlarged below. Intensity traces (offset white lines) are plotted at right. Scale bars are 10 mum. Colocalization coefficients are the combined results of 2-3 independent experiments ( n = 28-30 total fields/group).

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Experimental details: Figure 1. Characterization of SIDT2. (A) Lysosomes (Lys) were isolated from mouse brain homogenates (Hom), and analyzed by immunoblotting using polyclonal goat anti-SIDT2 antibody and antibodies against LAMP2 (lysosomal marker), RAB7A (late endosome and lysosome), RAB5A (early endosome), CANX (endoplasmic reticulum), COX4I1 (mitochondria), GOLGA1 (Golgi apparatus), GAPDH (cytosol), LMNA/lamin A (nuclei), and MAP1LC3A/B (autophagosome). (B) Neuro2a cells expressing GFP-tagged SIDT2 were incubated with LysoTracker Red. Fluorescence images were visualized using a confocal laser-scanning microscope. Scale bar: 10 mum. Colocalization rate was quantified using ImageJ software (right panel, n = 3). ( C ) Neuro2a cells expressing GFP-tagged SIDT2 were fixed, and immunostained using anti-RAB7A, anti-EEA1 (early endosomal marker) or anti-MAP1LC3A/B antibodies. Fluorescent images were obtained using confocal microscopy. Scale bars: 5 mum. Colocalization rate was quantified (right panels, n = 3). (D) Lysosomes were isolated from HeLa cells expressing SIDT2-FLAG or CTSB-FLAG. Isolated lysosomes (4 mug protein) were incubated with the indicated concentrations of trypsin at 37degC for 5 min. Proteins in the samples were analyzed by immunoblotting using an anti-FLAG antibody. (E) LAMP2C and SIDT2 or SIDT2-FLAG were overexpressed in HeLa cells. Cell lysates were prepared and immunoprecipitated with an anti-FLAG antibody. Cell lysates and the resulting immunoprecipitant were analyzed by immuno

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Experimental details: Figure 5 Altered trafficking of ATG9A in SMPD1 -deficient cells. (A) NAGLU activities ( top ), indicated immunoblots ( middle ) and ATG9A distribution profiles of density gradient fractions of postnuclear supernatant fractions from HC-3 and NPA-3 fibroblasts ( bottom ). When indicated, HC-3 fibroblasts were starved in HBSS plus 1 g/L glucose and treated with 2 nM ConA for 2 h (S/C). The migration of MAP1LC3B-I and MAP1LC3B-II are marked with a line and greater-than symbol, respectively. ATG9A distribution in fractions is expressed as percentage of the total ATG9A in all fractions as quantified by densitometry and ImageJ software. See figures S2A and B for additional data. (B and C) Representative confocal images of HC-1 and NPA-3 fibroblasts and CRISPr-Cas-9 wild-type (WT) and SMPD1 -KO (KO) MCF7 cells stained as indicated. When indicated, cells were starved in HBSS plus 1 g/L glucose for 2 h. Step size, 300 nm. See figures S2C and D for additional staining. (D) Manders coefficients for colocalization of TFRC with ATG9A and vice versa ( left ), and percentages of ATG9A and colocalized ATG9A-TFRC in the juxtanuclear area (4 um from nucleus) in WT and KO cells. Bars, SEM for 20 randomly chosen cells. (E) Representative immunoblots of the indicated proteins in lysates of WT and KO cells. (F) Quantification of the number of WIPI2 puncta/cell ( top ) and the percentage of ATG9A-positive WIPI2 puncta of all WIPI2 puncta ( bottom ) in WT and KO cells. Bars, SEM for 20 randomly chose

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Experimental details: FIGURE 1 Kir2.1 specifically binds to Golgin-97. Immunoprecipitation (IP) performed with (A) anti-Golgin-97; (C) anti-p230; (D) anti-GM130 antibodies from COS cells transfected with myc-Kir2.1 wild type (WT), the TGN-export mutant (Delta314-315), and the ER-export mutant (DeltaFCY) and then immunoblotted (IB) with myc-antibodies to detect Kir2.1 in the Golgin immunoprecipitates. 5% input is shown below; (B) Quantification of the relative amounts of Kir2.1 immunoprecipatated with Golgin-97. * P < 0.05.

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Experimental details: FIGURE 2 Golgin-97 GRIP domain directly interacts with Kir2.1. (A) Domain structure of Golgin-97, showing the GRIP domain and the coiled-coil domain. (B) Anti-myc immunoprecipitation (IP) followed by anti-HA immunoblotting (IB) from COS cells co-transfected with myc-Golgi 97 (Full length, Grip domain only, or coiled-coiled domain only) and HA-Kir2.1 wild type, WT, or the TGN-export mutant (Delta314-315) or (D) the HA-tagged Kir2.1 COOH-terminal cytoplasmic domain (CTCD). (C,E) Quantitative summaries comparing relative amounts of Kir2.1 (WT or Delta314-315) immunoprecipitated with full-length Golgin-97, or GRIP or Coiled-Coil domain, * P < 0.05. (F) GST pulldowns: GST alone or GST fusion proteins of the Kir2.1 CTCD were used to test for direct interaction with His-tagged GRIP domains of Golgin-97 or Golgin-245 purified from bacteria. Protein bound is shown relative to 5% input.

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Experimental details: FIGURE 4 Mapping the residues in GRIP domain involved in Kir2.1 binding. (A) Model of the Golgin-97 GRIP domain. First helix directly interacts with Arl1, while the third helix contains residues (M733, Y740) that are necessary for binding to Kir2.1. (B) The HA-tagged Kir2.1 channel was co-transfected with myc-tagged Golgin-97 (WT or GRIP domain mutants), and cell lysates were subjected to immunoprecipitation with anti-myc and then immunoblotted with anti-HA to detect Kir2.1 bound. (C) Summary Data, Relative to Average WT Kir2.1 immunoprecipitated with wild type (WT) Golgin-97 GRIP. * P < 0.05.

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Experimental details: FIGURE 5 Golgin-97 Interacts with Kir2.1 in the Golgi. (A) Confocal images of Kir2.1 (TGN-export mutant (Delta314-315) compared to ER-export mutant DeltaFCY) in COS-cells (green) and Golgi-97 (red) before (top) and after 10 min of brefeldin A treatment. (B) Anti-Golgin-97 immunoprecipitation (IP) followed by anti-HA immunoblotting (IB) from COS cells transfected with the HA-Kir2.1 mutants and treated with vehicle (-) or Brefeldin (+).

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Experimental details: Figure 6 B5 silencing does not block polyadenylation or nuclear export of RNA. (A) Transfected cells were infected with HSV-2(G) (moi 3 pfu/cell) and 3 h pi, cells were harvested and nuclear and cytoplasmic fractions isolated and Western blots were prepared and probed for golgin97, histone-1, B5 and ICP4. (B) Polyadenylated mRNA was isolated from the total RNA and from the cytoplasmic and nuclear fractions and then analyzed for ICP4 by qRT-PCR. Results are presented as the ratio of ICP4/RPLPO in each fraction (cytoplasmic or nuclear) to ICP4/RPLPO in the unfractionated total polyA RNA and are representative of 3 independent experiments.

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Experimental details: Figure 1 BARS interacts with LPAATdelta. ( a ) Representative confocal microscopy images of COS7 cells transfected with Flag-tagged LPAATgamma, LPAATdelta and LPAATeta, and fixed and processed for immunofluorescence with a monoclonal anti-Flag antibody (green) and with a polyclonal anti-TGN46 antibody (red; as indicated). Dotted lines indicate cell borders. ( b ) BARS immunoprecipitation (IP:BARS) of lysate from HeLa cells co-expressing BARS and LPAATgamma-Flag, LPAATdelta-Flag or LPAATeta-Flag. Representative western blotting (antibodies as indicated) of total lysate (input) and immunoprecipitated proteins with preimmune-IgG (Preim-IgG) or anti-BARS-IgG (as indicated). IgG H , IgG heavy chain. ( c ) Immunoprecipitation with an anti-Flag antibody (IP:Flag) of lysate from HeLa cells co-transfected with BARS and LPAATgamma-Flag, LPAATdelta-Flag or the empty vector. Representative western blotting of total lysate (input) and Flag-immunoprecipitated proteins with an anti-Flag monoclonal antibody or the anti-BARS polyclonal antibody (as indicated). ( d ) Representative electron microscopy image of HeLa cells transfected with Flag-tagged LPAATdelta for 24 h, and fixed and processed for cryo-immuno-electron microscopy with a monoclonal anti-Golgin-97 antibody (15-nm gold particles) and with a polyclonal anti-LPAATdelta antibody (10-nm gold particles). Molecular weight standards (kDa) in b and c , are indicated on the left of each panel. Data are representative of three independent e

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Experimental details: Figure 8 Trafficking of HPV16 to multivesicular endosomes requires CD63/syntenin-1 complex. ( a ) Representative pictures of CD63-GFP (green), LBPA (red), and HPV16 L1 (blue) triple staining with LBPA mAb and L1 pAb K75 antibodies. ( b,c ) Colocalisation analysis of HPV16 L1 and LBPA. HeLa cells were transfected with control siRNA, CD63 siRNA or syntenin-1 siRNA and infected with HPV16 PsV and stained with LBPA mAb and L1 pAb K75 antibodies. ( b ) Shows representative pictures of L1 (red) and LBPA (green) colocalisation. Nuclei are shown in blue. ( c ) Colocalisation of LBPA and L1 as well as early endosome marker EEA1 and L1 was analysed as in Fig. 3e ( P = 0,062 for CD63 siRNA and 0,069 for syntenin-1 siRNA). ( d,e ) Colocalisation analysis of viral DNA and the Golgi. HeLa cells were transfected with control, CD63 or syntenin-1 siRNA and infected with EdU-HPV16 PsV for 7 or 12 hours. ( d ) Shows representative pictures of viral DNA (vDNA, green) and Golgi (red) 12 hours post infection. ( e ) Colocalisation of viral DNA and Golgi was analysed as above and normalized to infected control siRNA treated cells. * P < 0.05 compared to control.

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Experimental details: Figure 4 (A) Isobolograms analysis of inhibition of growth of U87MG cells (A-C) and effects of RTV and IDV treatment on GLUT1/SLC2A1 transporter levels in U87MG cells (D-E). (A-C) The ""line of additivity"" connects the values of the IC50 of the two drugs used in combination, thus separating the quadrant into two parts; points plotted below the line of additivity indicate synergy (activity of the combination is larger than expected for the simple combination of the two drugs), whereas points above the line indicate antagonism (activity of the combination is lower than the expected for the simple combination of the two drugs). BCNU-RTV: (A) The two drugs at all tested concentrations show synergic activity. TMZ-RTV: (B) The two drugs at all tested concentrations show synergic activity. BCNU-IDV: (C) The two drugs show addictive or antagonistic behavior at all concentrations except for the highest, when we obtained an indication of minimal synergic activity. (D) Western blot of total lysates of U87MG cells: RTV reduced the level of GLUT1/SLC2A1 immunoreactivity, whereas IDV increased GLUT1/SLC2A1 level compared with untreated cells (NT). The same membrane was stripped and reacted with an antibody against actin (Act) whose level was similar in all samples. (E) As in D, but the cells were fractionated, and only the fraction containing the LDMs, where the GLUTs-containing vesicles reside, was analyzed by Western blot. As in the total extract, the GLUT1/SLC2A1 signal was decreased o

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Experimental details: Fig 2 Examination of GFP-G1 by confocal fluorescence microscopy shows that it accumulates and colocalizes with markers of the trans-Golgi network and endosomal recycling compartment. (A) Low magnification survey illustrating that GFP-G1 generally is accumulated perinuclearly, mostly in a finely granular pattern with occasional surrounding brighter puncta. (B-G) Higher magnification images comparing the distribution of GFP-G1 to organelle markers. (B) Minimal co-localization with insulin at the cell periphery where mature granules accumulate but noticeable overlap perinuclearly where storage granules are being formed. (C) Extensive overlap with proinsulin accumulated perinuclearly and resembling the perinuclear overlap seen in panel B. Expanded insets (below) show the GFP-G1 fluorescence has the same pattern as proinsulin but is not especially co-concentrated with proinsulin in brighter puncta of the latter. (D) GFP-G1 exhibits a highly similar pattern to the trans-Golgi/TGN tethering protein Golgin-97 but is more diffusely distributed. (E) Limited overlap of GFP-G1 with EEA1 marking early endosomes. (F) Limited overlap of GFP-G1 with NPC1 marking late endosomes and lysosomes. (G) Substantial overlap of GFP-G1 with transferrin receptor (TfR), which is concentrated in the endosomal recycling compartment. GFP-G1 is also visible at the plasma membrane and diffusely within the cytosol (presumably ER). (H) Coefficients determined by Manders' overlap analysis for each of the m

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Experimental details: Fig 4 ABCG1 and GFP-G1 exhibit rapid turnover. (A) Pulse-chase biosynthetic labeling with 35 S-amino acids and immunoadsorption of ABCG1. Phosphorimage (below) and quantification (above) show that the transporter in INS1 cells is short-lived with a halftime of turnover of ~2h, as in other cell types. Data are taken from one experiment (full phosphorimage shown in Figure A in S3 Fig ). (B) Results from western blotting showing loss of ABCG1 following inhibition of protein synthesis (by cycloheximide) occurs by both ERAD/proteasomal degradation (inhibited by MG132) and lysosomal degradation (inhibited by the acidification blocker bafilomycin A). Data are from 7 experiments; error bars indicate SEM. Results for individual experiments are in S1 File . (C) Representative fluorescence images from samples treated for 0, 2, and 6h with cycloheximide (CHX). Both diffuse cytoplasmic GFP-G1 (presumed to be ER associated) and GFP-G1 colocalized with the TGN marker Golgin-97 are lost by 2h while the remainder of accumulated perinuclear GFP-G1 and much of the cell surface transporter is lost by 6h. (D) Complementary images to panel C showing perinuclear GFP-G1 remains colocalized with TfR at 2h post cycloheximide addition but is lost by 6h. Images for panels C and D are taken from one of two independent experiments that were performed to evaluate the loss of GFP-G1 colocalized with both Golgin-97 and TfR. (E) Manders' overlap analysis for colocalization of GFP-G1 with Golgin-97 an

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Experimental details: YIPF6 localizes to the Golgi apparatus. Immunofluorescence of modified 22Rv1 cells stained with antibodies specific for YIPF6 (green) and Golgin-97 (red). pcDNA4 is 22Rv1 cells transfected with vector control. YIPA cells overexpress the full length variant of YIPF6, and the siYIPF6 represents 22Rv1 cells that have been treated with siRNA specific for YIPF6.

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Supportive validation

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Supportive validation

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Experimental details: Fig. 3 Compound 2a counteracts G93A-mediated Golgi fragmentation. a merged confocal stacks for GFP and Golgin97 in untransfected-; pcDNA3.1(+)SOD1 G93A (1 µg/well)-pCAAG-GFP (0.2 µg/well)-; pcDNA3.1(+)SOD1 G93A (1 µg/well)-pCAAG-GFP (0.2 µg/well) + 2a (10 µM)- and pCAAG-GFP (1.2 µg/well)-transfected Neuro2a (4 x 10 4 cells/well). Morphological analysis of Golgi in normal, intermediate or fragmented was done in nontransfected cells ( n = 83 independent cells), G93A-GFP ( n = 88 independent cells), G93A-GFP+ 2a ( n = 84 independent cells), and GFP ( n = 109 independent cells). The histogram in panel a shows Golgi subclasses (% of the total number of Golgi constructs, means +- SD) examined over four independent experiments, p < 0.0001 for all comparisons. Protein lysates from nontransfected and transfected Neuro2a cells (3 x 10 5 cells/well, pcDNA3.1(+)SOD1 G93A (2 µg/well)-pCAAG-GFP (0.5 µg/well) or with pCAAG-GFP (2 µg/well) were immunoblotted with antibodies against Golgin97 and beta-Actin. The histogram shows quantifications (mean fold changes +- SD versus untreated cells, n = 3 wells/group). b Neuro2a cell viabilities (mean ratio +- SD compared to untransfected cells, n = 19 independent wells) upon the following treatments: pCMV-LacZ (1.25 µg/well, n = 18 independent wells), pcDNA3.1(+)SOD1 G93A (1.25 µg/well, n = 18 independent wells), pcDNA3.1(+)SOD1 G93A (1.25 µg/well) + 2a (10 µM, n = 24 indep

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Experimental details: Fig. 1 Organelle perturbations associated with erythroid iron restriction. a Documentation of Golgi disruption by immunofluorescence on human CD34+ progenitors cultured 3 days in iron-replete (100% TSAT) or deficient (10% TSAT) erythroid medium +- isocitrate rescue (IC). Red: Golgin97; blue: DAPI. Graph: % cells with detectable Golgi. Numbers indicate mean +- SEM; n = 3 biologically independent experiments; ***, **** P = 0.002, 0.0001, one-way ANOVA with Tukey post hoc test. b Golgi assessment in human marrow samples by immunoperoxidase on clot sections from non-anemic controls and patients with anemia of chronic disease and inflammation (ACDI). 200x magnification. Brown: Giantin; blue: hematoxylin. Graph: % of marrow samples with Golgi disruption in >50% erythroblasts. n = 5 for control and 10 for ACDI patient samples; * P = 0.017, Fisher''s exact two-tailed test. c Effects on pericentriolar material (Pericentrin: PCTN) and adjacent Golgi documented by immunofluorescence on cells as in a . Red: Golgin97; green: Pericentrin; blue: DAPI Graph: % cells with PCTN+ centrosomes. Numbers indicate mean +- SEM; n = 3 independent experiments; *, ** P = 0.049, 0.037, one-way ANOVA with Tukey post hoc test. d Microtubule alterations demonstrated by immunofluorescence on cells as in a . Red: beta-tubulin; blue: DAPI. Graph: % cells with undetectable microtubule structures (MT). Numbers indicate mean +- SEM; n = 3 independent experiments; **** P = 0.0001, one-way ANOVA with Tukey post hoc

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Experimental details: Figure 2 TFG is essential for proper ULK1 complex distribution during autophagy A-D HeLa cells were transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR) and grown in fed or starved conditions for 1 h. Cells were fixed and co-labelled with ULK1 (red) antibody and (A) anti-CALNEXIN (green) antibody to highlight ER structures ( n = 3 independent experiments, n >= 16 fields analysed), (B) GOLGIN-97 (green) antibody to label GOLGI complex ( n = 3 independent experiments, n = 12 fields analysed), (C) SEC31A (green) to point out COPII vesicles ( n = 3 independent experiments, n >= 16 fields analysed) and (D) ERGIC53 (green) to mark ERGIC compartment ( n = 3 independent experiments, n >= 14 fields analysed). Analysis of ULK1 localization with each compartment is reported in each graph. Co-localization analyses were performed by ImageJ plugin Jacop. Values of Mander's coefficient for ULK1 are expressed in percentage as mean +- SEM. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. ** P < 0.01; *** P < 0.001; **** P < 0.0001; and ns, not significant. Scale bars 5 mum. White arrowheads point at ULK1 puncta associated with the analysed markers. E HeLa cells were transfected as in A), labelled with the indicated antibodies and imaged by SIM microscope. Scale bar in large panels, 5 mum; in small panels, 1 mum. N, nucleus. F HeLa cells were co-transfected with control siRNA and an

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Experimental details: Figure 4. Sterol-resistant SCAP overexpression induces the activation of NLRP3 in?ammasomes in VSMCs. (A) Representative immunoblots for NLRP3, Procaspase-1, cleaved caspase-1, IL-1beta, and IL-18 in SCAP-overexpressing VSMCs. (B) Expression of IL-1beta and IL-18 in VSMC supernatants as measured by ELISA. (C) Immunoblotting of immunoprecipitation with anti-SCAP or anti-NLRP3 in SCAP-overexpressing VSMCs. (D) Representative images of immuno?uorescence staining of NLRP3 (green) or SCAP (green) with Golgi(red) in control and SCAP-overexpressing VSMCs. (E) Representative immunoblot for NLRP3, Pro-caspase-1, cleaved caspase-1, IL-1beta, and IL-18 in SCAP-overexpressing VSMCs treated with lycorine for 4 h. (F) Expression of IL-1beta and IL-18 in VSMC supernatants as measured by ELISA. (G) Representative images of the immuno?uorescence staining of NLRP3 (green) or SCAP (green) with Golgi (red) in SCAP VSMCs after treatment with lycorine for 4 h. The data are presented as the means+-SD of 3 independent experiments. * P

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Experimental details: FIGURE 6 Golgin-97 is required for efficient delivery of Kir2.1 to the cell surface membrane from the Golgi. (A) Immunoblot of Golgin-97 from COS cells transfected with scramble or Golgi-97 siRNA. (B) Surface Expression of external HA-epitope tagged Kir2.1 in COS cells co-transfected with scramble or Golgi-97 siRNA or nothing. Mock are cells transfected with vector alone. (C) Representative confocal microscopy of Kir2.1 in COS cells transfected with scrample, or Golgin-97 siRNA. (D) Rescue Studies, Surface Expression of external HA-epitope tagged Kir2.1 in COS cells co-transfected with scramble or Golgi-97 siRNA and Golgin-97 rescue cDNA (WT vs mutant), * P < 0.05.

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Supportive validation

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Experimental details: Figure 1 Two-color STORM can be utilized to reveal spatial relationships of Golgi-localized proteins at high resolution. (A,B) Localization patterns of a cis -Golgi marker (GM130) and a trans -Golgi marker (Golgin-97) in the same COS7 cell visualized through conventional fluorescent microscopy (A) or through two-color STORM (B) . Scale bar, 500 nm. (C) A magnified view of the indicated area in (B) . Scale bar, 100 nm. (D) Localization patterns of a cis -Golgi marker (GM130) labeled with two different fluorescent dyes in the COS7 cell revealed through two-color STORM. Scale bar, 500 nm. (E) A magnified view of the indicated area in (D) . Scale bar, 100 nm. (F) Quantification of the percentage of overlapped pixels between GM130 and Golgin-97 and between Alexa Fluor 750-labeled GM130 and Alexa Fluor 647-labeled GM130 based on four super-resolution images acquired from one experiment in each experimental group (mean +- SD). *** p < 0.001 by two-tailed Student's t -test. The super-resolution images showed the localization patterns of the indicated protein in the whole juxtanuclear area. Each dot in the graphs represents data of the whole juxtanuclear Golgi area of each individual cell.

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Experimental details: FIGURE 4: Depletion of adaptor protein AP-1 results in BACE1 accumulation in the TGN. (A) HeLa cells stably expressing BACE1-GFP were transfected with either control siRNA, AP-1gamma siRNA, GGA1 siRNA, AP-4epsilon siRNA, or Arl5b siRNA for 72 h. (B) HeLa cells stably expressing Arl5b(Q70L)-GFP were transfected with either control siRNA or Arl5b siRNA for 72 h. Cells in A and B were lysed in RIPA buffer, and 20 mug of cell extracts was subjected to SDS-PAGE on 4-12% gradient polyacrylamide gel. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane and probed with (A) mouse anti-AP-1gamma, rabbit anti-GGA1, mouse anti-AP-4epsilon, (B) mouse anti-GFP, and (A and B) mouse anti-a-tubulin antibodies using a chemiluminescence detection system. (C) Monolayers of HeLa cells were fixed, permeabilized, and blocked, followed by staining with mouse anti-golgin-97 antibodies (red) and DAPI (blue). Higher magnification of the merged images of the cells marked * are also shown. Bar represents 10 mum. (D) The percentage of BACE1-GFP at the TGN was calculated as a percentage of total BACE1-GFP pixels that overlapped with golgin-97. Data are represented as the mean +- SD of three independent experiments ( n = 15) and analyzed by one-way ANOVA using Tukey's test. *** p < 0.001.

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Supportive validation

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Experimental details: APRIL associates with the Golgi apparatus. ( A ) APRIL is intracellularly localized. APRIL staining is shown in green, DNA staining with Topro-3 in red. ( B ) APRIL colocalizes with anti-human golgin-97 (CF4) monoclonal antibody, a marker for Golgi apparatus. APRIL staining is shown in green, staining for golgin-97 (CF4) in red. Yellow visualizes the overlap of the two fluorescent antibodies. Images are shown of transiently transfected 293T cells. Similar images were obtained on stably transfected cells. Asterisks indicate non-transfected cells serving as negative controls. Bar, 50 mum.

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Experimental details: Processing of APRIL takes place in the Golgi apparatus. ( A ) BFA abrogates cleavage of APRIL. HEK 293T cells were transiently transfected with APRIL, and treated for 5 h with BFA (3.5 muM), monensin (3.5 muM), chloromethylketone derivative (CMK; furin convertase inhibitor, 50 muM) or KB8301 (metalloprotease inhibitor, 10 muM) and subsequently analysed by western blot employing affinity-purified polyclonal anti-APRIL antibody. Proteins were separated in 15% SDS-PAGE. ( B ) Soluble APRIL cannot be immunoprecipitated from culture supernatants of BFA- or monensin-treated APRIL transfectants. Immunoprecipitates of culture supernatants from APRIL-transfected cells treated with BFA or monensin for 5 h were analysed by western blot. Proteins were separated on a 15% SDS-polyacrylamide gel. ( C ) Treatment with BFA, but not monensin, causes delocalization of APRIL. Cells were incubated with BFA (3.5 muM) or monensin (3.5 muM) for 5 h prior to fixing. Staining for golgin-97 or APRIL is shown in red, DNA staining with SybrGreen in green. Asterisks indicate non-transfected cells serving as negative controls. Bar, 10 mum.

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Experimental details: Fig. 2 Overexpressed trans-golgins form condensates. (A) HeLa cells were fixed and immunostained for endogenous golgins or (B) HeLa cells were transfected with golgin97-FLAG/Myc, golgin245-FLAG/Myc, GCC88-FLAG/Myc, or GCC185-FLAG/Myc, respectively, fixed 6 h post-transfection and immunostained for FLAG (exogenous golgin). Secondary antibody conjugated to Alexa Fluor 488 was used in (A) and (B). Each overexpressed golgin formed characteristic condensates. (C) Mean condensate area and (D) mean condensate circularity, where N denotes the number of condensates analyzed and error bars denote the standard error of the mean (SEM). The mean area (and SEM) was calculated based on condensates with area >0.8 um 2 , thereby excluding contributions from random fluctuations in protein concentration or fluorescence signal. The mean circularity (and SEM) was calculated by including condensates with area >1.5 um 2 because circularity became ill-defined for smaller particles. Golgin97 was excluded for the circularity analysis because too few condensates satisfied these criteria for accurate statistics. Scale bars: 5 um.