- MA3-067 - Invitrogen Antibodies RAB9A antibody

Submitted references Herpes Simplex Virus Entry by a Nonconventional Endocytic Pathway.

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Journal of virology 2020 Nov 23;94(24)

Endosomal trafficking defects in patient cells with KIAA1109 biallelic variants.

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Endocytosis of Activated Muscarinic m2 Receptor (m2R) in Live Mouse Hippocampal Neurons Occurs via a Clathrin-Dependent Pathway.

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Dynamic internalization and recycling of a metal ion transporter: Cu homeostasis and CTR1, the human Cu⁺ uptake system.

Clifford RJ, Maryon EB, Kaplan JH
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Affibody-mediated retention of the epidermal growth factor receptor in the secretory compartments leads to inhibition of phosphorylation in the kinase domain.

Vernet E, Lundberg E, Friedman M, Rigamonti N, Klausing S, Nygren PA, Gräslund T
New biotechnology 2009 Sep;25(6):417-23

Antibody-based proteomics for esophageal cancer: Identification of proteins in the nuclear factor-kappaB pathway and mitotic checkpoint.

Uemura N, Nakanishi Y, Kato H, Nagino M, Hirohashi S, Kondo T
Cancer science 2009 Sep;100(9):1612-22

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Cirillo C, Sarnelli G, Esposito G, Grosso M, Petruzzelli R, Izzo P, Calì G, D'Armiento FP, Rocco A, Nardone G, Iuvone T, Steardo L, Cuomo R
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High prevalence of retinoblastoma protein loss in triple-negative breast cancers and its association with a good prognosis in patients treated with adjuvant chemotherapy.

Treré D, Brighenti E, Donati G, Ceccarelli C, Santini D, Taffurelli M, Montanaro L, Derenzini M
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Coexpression of the melatonin receptor 1 and nestin in human breast cancer specimens.

Rögelsperger O, Ekmekcioglu C, Jäger W, Klimpfinger M, Königsberg R, Krenbek D, Sellner F, Thalhammer T
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Endocytosis of epithelial apical junctional proteins by a clathrin-mediated pathway into a unique storage compartment.

Ivanov AI, Nusrat A, Parkos CA
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Nitric oxide-dependent processing of heparan sulfate in recycling S-nitrosylated glypican-1 takes place in caveolin-1-containing endosomes.

Cheng F, Mani K, van den Born J, Ding K, Belting M, Fransson LA
The Journal of biological chemistry 2002 Nov 15;277(46):44431-9

Kaposi's sarcoma-associated herpesvirus K3 utilizes the ubiquitin-proteasome system in routing class major histocompatibility complexes to late endocytic compartments.

Lorenzo ME, Jung JU, Ploegh HL
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Quantitative analysis of the interactions between prenyl Rab9, GDP dissociation inhibitor-alpha, and guanine nucleotides.

Shapiro AD, Pfeffer SR
The Journal of biological chemistry 1995 May 12;270(19):11085-90

Quantitative analysis of the interactions between prenyl Rab9, GDP dissociation inhibitor-alpha, and guanine nucleotides.

Shapiro AD, Pfeffer SR
The Journal of biological chemistry 1995 May 12;270(19):11085-90

Rab GDI: a solubilizing and recycling factor for rab9 protein.

Soldati T, Riederer MA, Pfeffer SR
Molecular biology of the cell 1993 Apr;4(4):425-34

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of RAB9 was performed by loading 25 µg of K562 (lane 1), 293 (lane 2) and Hela (lane 3) onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4ºC overnight. The membrane was probed with a RAB9 monoclonal antibody (Product # MA3-067) at a dilution of 1:2000 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated secondary antibody for 1 hr at room temperature in the dark. Chemiluminescent detection was performed using Pierce ECL Plus Western Blotting Substrate (Product # 32132). Images were taken at an exposure time of 4 min (lane 1) and 2 min (lanes 2 and 3). Results show a band at ~23 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of RAB9 was achieved by transfecting HEK-293 cells with RAB9A specific siRNAs (Silencer® select Product # s17916, s17917). Western blot analysis (Fig. a) was performed using membrane enriched extracts from the HEK-293 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with RAB9 Mouse Monoclonal Antibody (Product # MA3-067, 1:2500 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 RAB9A.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-RAB9 Mouse Monoclonal Antibody (Product # MA3-067) and a 23 kDa band corresponding to RAB9 was observed in cell lines tested. Membrane enriched extracts (30 µg lysate) of HeLa (Lane 1), HEK-293 (Lane 2) and MCF7 (Lane 3) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0341BOX). 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 (1:2000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of RAB9 using RAB9 Monoclonal Antibody (Mab9) (Product # MA3-067) shows staining in A2058 Cells. RAB9 (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing RAB9 (Product # MA3-067) at a dilution of 1:100 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of RAB9 using RAB9 Monoclonal Antibody (Mab9) (Product # MA3-067) shows staining in Hela Cells. RAB9 (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing RAB9 (Product # MA3-067) at a dilution of 1:200 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of RAB9 using RAB9 Monoclonal Antibody (Mab9) (Product # MA3-067) shows staining in U251 Cells. RAB9 (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing RAB9 (Product # MA3-067) at a dilution of 1:100 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of RAB9 was performed using HEK-293 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 1 hour at room temperature. The cells were labeled with RAB9 Mouse Monoclonal Antibody (Product # MA3-067) at 1:200 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32723) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green) in HEK-293 cells. 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 of HEK-293 cells showing cytoplasmic (Golgi, ER, endosome pattern) and plasma membrane localization. Panel e 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: Immunohistochemistry was performed on cancer biopsies of deparaffinized Human prostate carcinoma tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:20 with a mouse monoclonal antibody recognizing RAB9 (Product # MA3-067) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on normal deparaffinized Human stomach tissue tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:20 with a mouse monoclonal antibody recognizing RAB9 (Product # MA3-067) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on normal deparaffinized Human tonsil tissue tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a mouse monoclonal antibody recognizing RAB9 (Product # MA3-067) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 2 Analysis of endosomal pathway at steady state in fibroblasts. Immunostaining for actin (magenta), lysosomal protein LAMP3 (red), and early endosome protein EEA1 (green) in patient B (Pt. B) and control (PCS Ctl) cells (A i-ii). Immunofluorescence imaging of patient B (Pt. B) and control (PCS Ctl) cells stained for actin (magenta), late endosome maker Rab9 (red), and early endosome protein EEA1 (green) (A iii-iv). Quantification of the average number of early endosomes (p = 8.49E-8) (B), lysosomes (p = 7.09E-28) (C), and late endosomes (not significantly different) (D) in fixed cell images from Pt. B and PCS Ctl cells. DNA counterstaining in blue (Hoechst). Error bars represent SEM. ** p < 0.01 * = p < 0.025. Fig. 2

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 4 Dextran endocytosis trafficking assay. Dextran overall intensity within the cell at 5 and 10 min of dextran chase in patient B (Pt. B) and control (PCS Ctl) cells (A). Correlation of dextran signal with EEA1 signal in cells at 5 and 10 min of dextran chase in Pt. B and PCS Ctl (B). Representative micrographs of immunofluorescence staining of dextran (green) labeled Pt. B and PCS Ctl cells at 5 min with various endosomal markers: Rab9 late endosomes (red) and EEA1 early endosomes (magenta) (C i-ii), Rab11 fast- (red) and Rab4 slow-recycling endosomes (C iii-iv), and Rab9 late endosomes (red) with Rab7 lysosomal targeted vesicles (magenta) (C v-vi). DNA counterstaining in blue (Hoeschst) (C). Correlation of each endosomal and trafficking marker with dextran signal at 0, 5 and 10 min chase time points (t0, t5 and t10, respectively) in dextran assay is shown as indicated in the vertical axis label (D-G). Error bars represent SEM. ** = p < 0.01, * = p < 0.025. Fig. 4

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: FIGURE 9 Immunohistochemical localization of m2R in neuronal compartments involved in endocytosis, synthesis, maturation, and degradation in fixed hippocampal neurons. Hippocampal neurons were transfected with m2R-WT. Neurons were stimulated with CCh at 30 muM for 6, 20 min and 1 h fixed, and processed for visualization of m2R together with markers of intraneuronal compartments and observed by confocal microscopy. ( A-C) 6 min after CCh stimulation (30 muM), some m2R immunopositive punta colocalize with CHC in clathrin-coated pits, EEA1 in early endosomes and Rab9 in late endosomes (arrow heads). (D-E) Twenty minutes after CCh stimulation (30 muM), we failed to detect no colocalization of m2R with PDI, a marker of endoplasmic reticulum and GM130, a marker of Golgi apparatus. (F-F) One hour after CCh stimulation (30 muM), some m2R immunopositive puncta colocalize with CathD, a marker of lysosomes (arrow heads). The quantitative analysis of the colocalization of m2R and markers of subcellular compartment in neurons was performed using the Jacop Plugin of ImageJ and statistical data are reported from the Costes's randomization-based colocalization module (see Materials and Methods). Data are expressed as a Pearson's coefficient (pc) and pc were compared using the Mann-Whitney U -test. Our analysis shows that the colocalization of the immunofluorescent signals for m2R with CHC, EEA1, Rab9, and CathD is higher after treatment with CCh compared to untreated neurons (CHC, Rab9

MA3-067

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