PA1-062

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of HeLa (Lane 1), HEK 293 (Lane 2), K-562 (Lane 3), SH-SY5Y (Lane 4) and 10uL conditioned media from HeLa cell line (Lane 5). The blot was probed with Anti-Furin Convertase Rabbit Polyclonal Antibody (Product # PA1-062, 1:500-1:2000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 87 kDa band corresponding to Furin Convertase was observed across the cell lines and conditioned media tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody using iBind™ Flex Western Starter Kit (Product # SLF2000S). Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: CRISPR-Cas9 mediated genome editing ofFURIN (as confirmed by next generation sequencing) was achieved by using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR694124_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Fig (a) Western blot analysis of FURIN was performed by loading 30 µg of HeLa wild type (Lane 1), HeLa Cas9 (Lane 2) and HeLa Cas9 cells transduced with FURIN Lentiviral sgRNA (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 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 Anti-Furin Polyclonal Antibody (Product # PA1-062) using 1:1000 dilution and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036 1:5000 dilution).Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). A loss of signal in sgRNA transduced cells using the LentiArray™ CRISPR product line confirms that antibody is specific toFURIN (Fig (b)).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Furin Polyclonal Antibody (Product # PA1-062) and a 87 kDa band corresponding to Furin was observed across all cell lines and tissues tested except Daudi and Ramos which are known to be low expressing cell lines (DOI: 10.1099/0022-1317-75-10-2821). Whole cell extracts (30 µg lysate) of HEK-293 (Lane 1), HaCaT (Lane 2), BeWo (Lane 3), SK-O-V3 (Lane 4), Daudi (Lane 5), Ramos (Lane 6), Mouse Liver (Lane 7) and Rat Liver (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-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Expression of Furin was observed in HEK-293, HaCaT, BeWo, SK-O-V3, Mouse Liver and Rat Liver but not in Daudi and Ramos which are known to be low expressing cell lines (DOI: 10.1099/0022-1317-75-10-2821).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunolocalization of endogenous furin in mouse 3T3 cells with PA1-062.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Furin was performed using 70% confluent log phase BeWo 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 Furin Polyclonal Antibody (Product # PA1-062) at 2 µg/mL dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (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). Panel d represents the merged image showing predominantly Golgi Complex and cytoplasmic localization. Panel e represents Daudi cells with lower expression of Furin (DOI: 10.1099/0022-1317-75-10-2821). Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Furin Convertase was performed using 70% confluent log phase HeLa cells. The cells were fixed with Methanol for 5 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with Furin Convertase Rabbit Polyclonal Antibody (Product # PA1-062) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Cytoskeleton was stained with alpha-Tubulin Monoclonal Antibody (Product # 32-2500, 1 µg/mL) followed by Goat anti-Mouse IgG Secondary Antibody, Alexa Fluor® 594 conjugate (Product # A-11032, 1:400) (Panel c: red). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel d represents the merged image showing cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 4 Furin internalization in hypoxic cells involves both clathrin and lipid rafts. Cells were seeded on collagen type IV-coated slides, cultured in normoxic or hypoxic chambers for 4 h. A : Micrographs of furin (eGFP), clathrin (red), and merged images. B : Micrographs of furin (eGFP), caveolin-1 (red), and merged images. C : Co-immunoprecipitation of furin with clathrin and caveolin-1 in HT-1080 cells incubated in normoxia or hypoxia. D : Graph showing the percentage of furin with the basal plasma membrane. Cells were seeded on collagen type IV-coated slides, left either untreated (control; CTL), or pretreated with MbetaCD (300 uM) or chlorpromazine (5 uM) prior to culture in normoxic or hypoxic chambers for 4 h. Cells were then labeled with DiI processed for confocal microscopy and fluorogram analysis were performed as described under Materials and Methods section. Column, mean; bar, SE; BPM, basal plasma membrane; * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 6 Filamin-A is essential for hypoxia-driven relocalization of furin to the plasma membrane. Cells were seeded on collagen type IV-coated slides and cultured in normoxic or hypoxic chambers for 4 h. A : Micrographs of furin (eGFP), filamin-A (red), and merged images. B : Co-immunoprecipitation of furin and filamin-A in normoxic or hypoxic cells. C : Graph showing the percentage of furin with the basal plasma membrane. HT-1080-eGFP-fur and HT-1080-eGFP-fur-VA 750 were seeded on top of collagen IV-coated slides and cultured either in normoxic or hypoxic chambers for 4 h and labeled with DiI. Fluorogram analysis were performed as described under Materials and Methods section. Column, mean; bar, SE; BPM, basal plasma membrane; *** P < 0.0007; scale bars correspond to 5 um.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 Overexpression of furin cDNA increases the expression level of 9F5 antigen. COS-7 cells were cotransfected with pcDNA3.1- Gpnmb /pcDNA3.1 or pcDNA3.1- Gpnmb /pcDNA3.1-furin vector. A : At 72 hr after transfection, cells were double-stained with 9F5 (red) and anti-GPNMB (green) antibodies. B : The results shown in A were quantified and are given as means +- SEM ( n = 3). Fluorescence intensities (9F5 and anti-GPNMB) in cells transfected with pcDNA3.1- Gpnmb /pcDNA3.1were set at 100%. * P < 0.05 by Dunnett's multiple comparison test. C : Cell lysates were also subjected to immunoblot analysis for GPNMB, furin, and beta-actin. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com .]

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 9 A furin siRNA inhibits the expression level of 9F5 antigen. HEK293 cells were cotransfected with pcDNA3.1- Gpnmb /control small interfering RNA (siRNA) or pcDNA3.1- Gpnmb /furin siRNA. A : At 72 hr after transfection, cells were double-stained with 9F5 (red) and anti-GPNMB (green) antibodies. B : The results shown in A were quantified and are given as means +- SEM ( n = 3-4). Fluorescence intensities (9F5 and anti-GPNMB) in cells transfected with pcDNA3.1- Gpnmb /pcDNA3.1 were set at 100%. ** P < 0.01 by Dunnett's multiple comparison test. C : Cell lysates were also subjected to immunoblot analysis for furin and beta-actin. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com .]

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 10 IL-12 increases both furin and 9F5 antigen of rat type 1 MG with similar kinetics. Rat type 1 MG were treated with rat recombinant IL-12 (10 ng ml -1 ) at the indicated hours, and cell lysates were subjected to immunoblot analysis for 9F5 antigen, furin, GPNMB, and beta-actin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIG 2 Growth kinetics and maturation status of DENVs on Vero and VF1 cells. (A) Schematic of the sleeping beauty-based transposon cassette for ectopic expression of human furin (hFurin). A bidirectional EF1a promoter was used to drive the expression of hFurin and red fluorescent protein (RFP) with a puromycin resistance gene (Puro) linked by a 2A self-cleaving peptide (P2A). (B and C) Western blot (B) and immunofluorescence (C) images of polyclonal and clonal VF1 using anti-furin antibodies. (D to G) Growth kinetics and degree of maturation of DENV1-WT (D), DENV2-WT (E), DENV3-WT (F), and DENV4-WT (G) in unmodified Vero cells (black circles) and VF1 cells (blue triangles). Cells were infected with DENV at an MOI of 0.01 to 0.05 for 120 h. Supernatants were harvested every 24 h for 120 h, and the 120-h supernatants were analyzed by Western blotting for DENV maturation using anti-Env and anti-prM antibodies. All assays were performed with at least two biological repeats with two technical replicates. Growth kinetics of DENV variants were compared to their corresponding wild type using two-way ANOVA multiple comparisons.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 1 Starvation translocates TGN membrane proteins to endosomes. All cells are HeLa cells. a , b Furin loses its Golgi localization during starvation. Cells treated with indicated medium for 1 h and endogenous furin and Golgin-245 were stained. The fraction of Golgi-localized furin is quantified in b . c The recovery of Golgi localization of furin after supplying nutrient. After starvation in HBSS for 2 h, cells were treated with DMEM for indicated time and stained as in a . d Kinetics of Golgi-localized furin-GFP during HBSS and subsequent DMEM treatment. Cells expressing furin-GFP were first starved in HBSS for 2 h and subsequently stimulated by DMEM for 2 h. At indicated time, cells were stained for endogenous Giantin and the fraction of Golgi-localized furin-GFP is quantified. e , f Nutrient starvation significantly reduces the Golgi localization of CD8a-furin. Cells transiently expressing CD8a-furin were treated by indicated medium for 2 h and stained as in e . The fraction of Golgi-localized CD8a-furin is quantified in f . g The translocation of CD8a-furin to the endosome during nutrient starvation. Cells transiently expressing indicated constructs were treated with HBSS for 2 h and stained. Boxed regions are enlarged at the upper right corner. Arrows indicate colocalization. h , i The endosomal pool of CD8a-furin increases during nutrient starvation. Similar results have been observed in four independent experiments. Cells stably expressing CD8a-fu

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 TGF-beta1 upregulates the expression of furin in human granulosa-lutein cells. ( A , B ) SVOG cells were treated with vehicle control (PBS) or various concentrations (0.1, 1, or 10 ng/mL) of TGF-beta1 for 12 h ( A ) and 24 h ( B ), and the mRNA ( A ) and protein ( B ) levels of furin were examined using RT-PCR ( A ) and Western blot ( B ), respectively. ( C , D ) SVOG cells were treated with 1 ng/mL TGF-beta1 for 3, 6, 12 or 24 h, and the mRNA ( C ) and protein ( D ) levels of furin were examined using RT-PCR ( C ) and Western blot analysis ( D ), respectively. ( E ) hGL cells were treated with vehicle control or various concentrations (0.1, 1, or 10 ng/mL) of TGF-beta1 for 12 h, and the mRNA levels of furin were examined using RT-PCR. The results are expressed as the mean +- SE of at least three independent experiments. Values with different lower-case letters are significantly different ( p < 0.05). If a pair of values is significantly different ( p < 0.05), the values have different subscript letters (a vs. b or b vs. c) assigned to them.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 The SMAD3-SMAD4 signaling pathway is required for the TGF-beta1-induced upregulation of GDNF and furin in SVOG cells. ( A ) SVOG cells were transfected with 25 nM siCtrl, 25 nM siSMAD2 25 nM siSMAD3 or 25 nM siSMAD4 for 48 h. The mRNA levels of SMAD2, SMAD3 and SMAD4 were examined using RT-qPCR. ( B , C ) SVOG cells were transfected for 48 h with 25 nM siCtrl, 25 nM siSMAD2, 25 nM siSMAD3 or 25 nM siSMAD4 and then treated with the vehicle control or 1 ng/mL TGF-beta1 for an additional 12 h. The mRNA levels of GDNF ( B ) and furin ( C ) were examined using RT-qPCR. ( D ) SVOG cells were transfected for 48 h with 25 nM siCtrl, 25 nM siSMAD2, 25 nM siSMAD3 or 25 nM siSMAD4 and then treated with the vehicle control or 1 ng/mL TGF-beta1 for an additional 24 h. The protein levels of GDNF, furin, SMAD2, SMAD3 and SMAD4 were examined using Western blot analysis. The results are expressed as the mean +- SE. Values with different lower-case letters are significantly different ( p < 0.05). If a pair of values is significantly different ( p < 0.05), the values have different subscript letters (a vs. b or b vs. c) assigned to them.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 AP-1A is necessary for furin localization at the TGN. mu1A -/- fibroblasts stably transfected with mu1B alone (top) or with mu1B and mu1A-HA (bottom) were double-labeled with anti-gamma-adaptin antibodies (left) and antifurin antibodies (middle) in combination with Alexa 488-labeled (anti-gamma staining) and Alexa 594-labeled (antifurin staining) secondary antibodies. Specimens were analyzed by confocal microscopy and representative images are shown. The arrows denote regions in the AP-1B/AP-1A-HA transfectants that are positive for AP-1 but negative for furin.