anti rab5 primary antibody  (Cell Signaling Technology Inc)

Journal: The Journal of Biological Chemistry

Article Title: Rab5 nucleotide binding promotes oxidative metabolism to fuel hepatocellular carcinoma cell proliferation

doi: 10.1016/j.jbc.2026.111321

Figure Lengend Snippet: Rab5 is recruited to lipid droplets (LDs) in a GTPase-dependent manner in Hep3B cells . Super-resolution confocal micrographs of Hep3B human hepatoma cells expressing ( A ) WT Rab5 (GFP-Rab5 WT), ( B ) constitutively active mutant (GFP-Rab5 Q79L), and ( C ) dominant-negative mutant (GFP-Rab5 S34N). LDs were stained with Oil Red O (ORO, red ) to visualize neutral lipid stores. GFP-Rab5 fluorescence (green ) marks Rab5 localization. Inlays depict Rab5 localization around LD borders, indicated further by yellow arrows . D , quantification of Rab5–LD colocalization by the ratio of LD:cytoplasm GFP-Rab5 intensity. Graphs represent mean ± SD from n = 3 independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. ∗∗ p < 0.01 and ∗∗∗ p < 0.001.

Article Snippet: Formalin-fixed, paraffin-embedded sections were stained with an anti-Rab5 primary antibody (Cell Signaling Technology, 3547) followed by standard immunohistochemistry detection procedures.

Techniques: Expressing, Mutagenesis, Dominant Negative Mutation, Staining, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Rab5 nucleotide binding promotes oxidative metabolism to fuel hepatocellular carcinoma cell proliferation

doi: 10.1016/j.jbc.2026.111321

Figure Lengend Snippet: Enhanced recruitment of Rab5 to lipid droplets (LDs) upon lipophagy stimulation . A , Western blot analysis of Rab5 and EEA1 GTP-pulldown in regular medium (CT) or 4 h HBSS starvation in Hep3B cells. B , quantification of the fold change of GTP-bound/total Rab5 as well as EEA1 from n = 4 independent experiments. C , quantification of total Rab5–β-actin confirming that starvation does not change overall Rab5 expression. D , Western blot analysis of LDs isolated from Hep3B cells by density gradient centrifugation showing abundant levels of Rab5 in isolated LDs following 4 h of HBSS starvation compared with the regular medium (CT). E , quantification of Rab5–Plin2 in the biochemically isolated LDs. F , fluorescence micrograph showing immunofluorescence staining of Hep3B cells expressing Myc-tagged Rab5 ( green ). Cells were treated with oleic acid for 16 h and then either maintained in regular medium (control) or subjected to 4 h of HBSS starvation before being fixed and stained with Oil Red O (ORO; red ) to label LDs. Note the marked increase in Rab5 staining around LDs in HBSS-starved cells versus control ( yellow arrowheads ). Graphs represent mean ± SD from n = 3 to 4 independent experiments. Statistical significance was determined using an unpaired two-tailed t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. CT, control; EEA1, early endosome antigen 1; HBSS, Hank’s balanced salt solution; Plin2, perilipin-2.

Article Snippet: Formalin-fixed, paraffin-embedded sections were stained with an anti-Rab5 primary antibody (Cell Signaling Technology, 3547) followed by standard immunohistochemistry detection procedures.

Techniques: Western Blot, Expressing, Isolation, Gradient Centrifugation, Fluorescence, Immunofluorescence, Staining, Control, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Rab5 nucleotide binding promotes oxidative metabolism to fuel hepatocellular carcinoma cell proliferation

doi: 10.1016/j.jbc.2026.111321

Figure Lengend Snippet: Inhibition of Rab5 GTPase activity reduces lipid droplet (LD) catabolism via its GTPase activity . A , Western blot analysis of Rab5 and EEA1 GTP-pulldown in Hep3B cells treated with DMSO (CT) or NAP (100 μM, 48 h). B , quantification of GTP/total Rab5 as well as EEA1 from n = 3 independent experiments. C , confocal micrograph showing ORO-stained LDs ( red ) and DAPI-stained nuclei from Hep3B cells treated with DMSO (control) and NAP (100 μM). D , bar graphs depict quantification of LD number/cell, total LD area/cell, and LD size. E , confocal micrograph showing ORO-stained LDs (red ) and DAPI-stained nucleus from Hep3B cells treated with DMSO (control) and NAP (100 μM) with oleic acid (OA, 150 μM). F , bar graphs depict quantification of LD number/cell, total LD area/cell, and LD size. G , cartoon depicting NAP alteration of Rab5 GTP binding. Graphs represent mean ± SD from n = 3 independent experiments. Statistical significance was determined using an unpaired two-tailed t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. CT, control; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; EEA1, early endosome antigen 1; NAP, neoandrographolide; ORO, Oil Red O.

Article Snippet: Formalin-fixed, paraffin-embedded sections were stained with an anti-Rab5 primary antibody (Cell Signaling Technology, 3547) followed by standard immunohistochemistry detection procedures.

Techniques: Inhibition, Activity Assay, Western Blot, Staining, Control, Binding Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Rab5 nucleotide binding promotes oxidative metabolism to fuel hepatocellular carcinoma cell proliferation

doi: 10.1016/j.jbc.2026.111321

Figure Lengend Snippet: Inhibition of Rab5 decreases HCC energy homeostasis . A , Seahorse metabolic analysis showing NAP (100 μM, 48 h) inhibition of Rab5 decreases oxygen consumption rate (OCR) compared with control in complete media. n = 3 independent experiments. B , quantification of basal and spare respiratory capacity in complete media following treatment with NAP. C , Seahorse metabolic analysis showing DMSO versus NAP treatment in Hep3B cells under 4 h HBSS starvation. D , quantification of basal and spare respiratory capacity from C . Graphs represent mean ± SD from n = 3 independent experiments. Statistical significance was determined using an unpaired two-tailed t test. ∗ p < 0.05, ∗∗ p < 0.01. DMSO, dimethyl sulfoxide; HBSS, Hank’s balanced salt solution; HCC, hepatocellular carcinoma; NAP, neoandrographolide.

Article Snippet: Formalin-fixed, paraffin-embedded sections were stained with an anti-Rab5 primary antibody (Cell Signaling Technology, 3547) followed by standard immunohistochemistry detection procedures.

Techniques: Inhibition, Control, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Rab5 nucleotide binding promotes oxidative metabolism to fuel hepatocellular carcinoma cell proliferation

doi: 10.1016/j.jbc.2026.111321

Figure Lengend Snippet: Rab5 is upregulated in HCC and associated with cancer hallmarks . A , volcano plot from TNMplot showing expression of known lipid droplet (LD)–associated proteins, including Rab5A/B/C, in HCC tumors versus adjacent normal tissues from n = 53 patients. B , representative immunohistochemistry images from a liver tissue microarray showing Rab5 staining in normal liver (n = 4) versus HCC specimens (n = 7). C , quantification of Rab5 immunohistochemistry scores from the tissue microarray (mean ± SD, ∗∗∗ p < 0.0002; Welch's t test). D , cancer hallmark gene set enrichment analysis based on the study by Menyhart et al . , showing Rab5-associated genes enriched in proliferation, invasion, and evading growth suppressors. E , Kaplan–Meier survival analysis from the TCGA–LIHC dataset stratified by Rab5 expression. High Rab5 expression correlates with poorer overall survival (log-rank test, p = 0.06). HCC, hepatocellular carcinoma; LIHC, Liver Hepatocellular Carcinoma Cohort; TCGA, The Cancer Genome Atlas.

Article Snippet: Formalin-fixed, paraffin-embedded sections were stained with an anti-Rab5 primary antibody (Cell Signaling Technology, 3547) followed by standard immunohistochemistry detection procedures.

Techniques: Expressing, Immunohistochemistry, Microarray, Staining

Journal: The Journal of Biological Chemistry

Article Title: Rab5 nucleotide binding promotes oxidative metabolism to fuel hepatocellular carcinoma cell proliferation

doi: 10.1016/j.jbc.2026.111321

Figure Lengend Snippet: Working model depicting Rab5-mediated lipid droplet (LD) catabolism in HCC . Under nutrient deprivation, Rab5 GTP binding is increased and promotes a higher frequency of interaction with LDs, driving tethering and fusion with lysosomes via microlipophagy. Inside the lysosome, lysosomal acid lipase (LAL) degrades LD, releasing free fatty acids (FFAs), which are then shuttled to mitochondria for β-oxidation to fuel HCC cell proliferation and survival. HCC, hepatocellular carcinoma.

Article Snippet: Formalin-fixed, paraffin-embedded sections were stained with an anti-Rab5 primary antibody (Cell Signaling Technology, 3547) followed by standard immunohistochemistry detection procedures.

Techniques: Binding Assay

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A and B ) Affibody-chase experiments. Cells surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP) to stimulate α V β 6 integrin and trigger α V β 6 endocytosis, or vehicle (Control), 0- to 60-min time course. Quantitation represents cytoplasmic HER2 fluorescence intensity analysis in (A) trastuzumab-sensitive or (B) trastuzumab-resistant BT474 cells ( N = 3; 27 to 50 cells per condition), normalized to control trastuzumab-sensitive BT474 cells (0 min); scale bar, 10 μm. Two-way ANOVA with Šídák’s multiple comparison test. Image intensity increased in (B), relative to (A), due to low cell surface HER2 levels in trastuzumab-resistant cells to highlight internalization differences. ( C ) HER2 (green) and RAB5 (magenta) immunofluorescence in trastuzumab-sensitive and trastuzumab-resistant BT474 cells, treated with soluble LAP, 0 to 60 min ( N = 3; 16 to 28 cells per condition); scale bar, 10 μm. ( Ca ) HER2/RAB5 colocalization quantitation (Pearson’s coefficient ± SEM). Two-way ANOVA with Dunnett’s multiple comparison test. ( D ) Active RAB5 pull-down assays. 0- to 60-min LAP stimulation time course. Quantitation of mean RAB5 activity (pull-down eluate), relative to total RAB5 (lysate) ± SEM ( N = 3), normalized to 0-min trastuzumab-sensitive cells. One-way ANOVA with Dunnett’s multiple comparison test. ( E and F ) Affibody-chase experiments in (E) siControl Trastuzumab-Sensitive or (F) Trastuzumab-Resistant BT474 cells expressing constitutively active RAB5 (RAB5CA), dominant-negative RAB5 (RAB5DN), dominant-negative RAB7 (RAB7DN), or mCherry vector control. Cells were surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP), or vehicle control (control), for 0 or 30 min. Quantitation represents cytoplasmic HER2 fluorescence intensity ( N = 3; 81 to 87 cells per condition); scale bar, 10 μm. One-way ANOVA with Tukey’s multiple comparison test. Representative images in fig. S10 (A and B). Further HER2 internalization analyses: Supplementary Results and fig. S11 (A to D). [(A), (B), and (D) to (F)] Data are arbitrary units (AU) normalized to control means ± SEM. [(A) to (F)] Statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Primary antibodies against RAB5 (Cell Signaling Technology, rabbit mAb #3547), RAB7 (Cell Signaling Technology, rabbit mAb #9367), GDI2 (Thermo Fisher Scientific, rabbit pAb #pa5-48831), HER2 (Cell Signaling Technology, rabbit mAb #2165), pHER2 Y877 (Abcam, rabbit mAb #2241), pHER2 Y1248 (Abcam, rabbit mAb #2247), pHER2 Y1222 (Abcam, rabbit mAb #2243), pHER2 Y1196 (Abcam, rabbit mAb #6942), pHER2 Y1112 (Millipore, mouse mAb #04-294), αV-integrin (Abcam, rabbit mAb #ab179475), β6-integrin (Santa Cruz Biotechnology, goat pAb #sc-6632), β1-integrin (Abcam, rabbit mAb #ab52971), vinculin (Abcam, mouse mAb #ab11194), paxillin (BD, mouse mAb #610051), phospho-Erk 1/2 (p44/42 MAPK) T202/Y204 (pERK1/2) (Cell Signaling Technology, rabbit mAb #137F5), Akt (Cell Signaling Technology, rabbit mAb #4691), pAkt (Cell Signaling Technology, rabbit mAb #4060), GAPDH (Abcam, mouse mAb #ab9484), or β-actin (Sigma-Aldrich, mouse mAb #A3853) were incubated at 4°C overnight.

Techniques: Labeling, Control, Quantitation Assay, Fluorescence, Comparison, Immunofluorescence, Activity Assay, Expressing, Dominant Negative Mutation, Plasmid Preparation

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A and B ) Affibody-chase experiments: siControl-transfected or siGDI2-transfected BT474 cells surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP) to stimulate α V β 6 integrin and trigger α V β 6 endocytosis, or vehicle (Control), 0- to 60-min time course. Quantitation represents cytoplasmic HER2 fluorescence intensity analysis in (A) trastuzumab-sensitive or (B) trastuzumab-resistant BT474 cells ( N = 3; 74 to 160 cells per condition); scale bars, 10 μm. Two-way ANOVA with Tukey’s multiple comparison test. Image intensity increased in (B), relative to (A), due to low cell surface HER2 levels in trastuzumab-resistant cells to highlight internalization differences. ( C ) GDI2 (green) and RAB5 (magenta) immunofluorescence in trastuzumab-sensitive and trastuzumab-resistant BT474 cells ( N = 3; >120 cells per condition); scale bars, 5 μm. GDI2/RAB5 colocalization quantitation (Pearson’s coefficient ± SEM), two-sided t test. ( D ) Role of GDI2 in α V β 6 -dependent RAB5 activity modulation. Trastuzumab-sensitive and trastuzumab-resistant BT474 cells transfected with siRNA against GDI2 (siGDI2 #1 and #2) or control siRNA. 0- to 60-min LAP stimulation time course. Quantitation of mean RAB5 activity (pull-down eluate), relative to total RAB5 (lysate) ± SEM ( N = 3), normalized to 0-min trastuzumab-sensitive cells. N = 4 independent replicate experiments. Two-way ANOVA with Šídák’s multiple comparison tests. ( E ) Haptotactic migration analysis of BT474 cells (Trastuzumab-Sensitive and Trastuzumab-Resistant) in Transwell coated with FN or BSA as a negative control. Cells were transfected with siRNA against GDI2 (siGDI2 #1 and #2) or siRNA control. Migration was assessed over 24 hours in the presence or absence of α V β 6 integrin blocking antibody or trastuzumab. Data shown are means ± SEM ( N = 3). One-way ANOVA with Šídák’s multiple comparison tests. [(A), (B), (D), and (E)] Data are arbitrary units (AU) normalized to control means ± SEM. [(A) to (E)] Statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Primary antibodies against RAB5 (Cell Signaling Technology, rabbit mAb #3547), RAB7 (Cell Signaling Technology, rabbit mAb #9367), GDI2 (Thermo Fisher Scientific, rabbit pAb #pa5-48831), HER2 (Cell Signaling Technology, rabbit mAb #2165), pHER2 Y877 (Abcam, rabbit mAb #2241), pHER2 Y1248 (Abcam, rabbit mAb #2247), pHER2 Y1222 (Abcam, rabbit mAb #2243), pHER2 Y1196 (Abcam, rabbit mAb #6942), pHER2 Y1112 (Millipore, mouse mAb #04-294), αV-integrin (Abcam, rabbit mAb #ab179475), β6-integrin (Santa Cruz Biotechnology, goat pAb #sc-6632), β1-integrin (Abcam, rabbit mAb #ab52971), vinculin (Abcam, mouse mAb #ab11194), paxillin (BD, mouse mAb #610051), phospho-Erk 1/2 (p44/42 MAPK) T202/Y204 (pERK1/2) (Cell Signaling Technology, rabbit mAb #137F5), Akt (Cell Signaling Technology, rabbit mAb #4691), pAkt (Cell Signaling Technology, rabbit mAb #4060), GAPDH (Abcam, mouse mAb #ab9484), or β-actin (Sigma-Aldrich, mouse mAb #A3853) were incubated at 4°C overnight.

Techniques: Transfection, Labeling, Control, Quantitation Assay, Fluorescence, Comparison, Immunofluorescence, Activity Assay, Migration, Negative Control, Blocking Assay

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A ) Trastuzumab-Sensitive Cells: GDI2 is recruited to sites proximal to α V β 6 IACs and coordinates HER2 and α V β 6 trafficking and signaling by locally modulating RAB5 activity. GDI2-mediated cross-talk between α V β 6 and HER2 affects membrane availability of both receptors, ultimately influencing migration, invasion, and TGFβ activation. ( B ) Trastuzumab-Resistant Cells: GDI2 is excluded from α V β 6 IACs, leading to dysregulation of RAB5 activation dynamics, followed by increased RAB7 activation. Consequently, HER2/α V β 6 cross-talk is impaired, altering receptor trafficking dynamics and disrupting bioavailability of both HER2 and α V β 6 integrin at the plasma membrane. This dysregulation further affects TGFβ activation, resulting in increased cell invasiveness and metastatic potential. Overall, these changes may increase the ability of cells to evade HER2 targeting drugs.

Article Snippet: Primary antibodies against RAB5 (Cell Signaling Technology, rabbit mAb #3547), RAB7 (Cell Signaling Technology, rabbit mAb #9367), GDI2 (Thermo Fisher Scientific, rabbit pAb #pa5-48831), HER2 (Cell Signaling Technology, rabbit mAb #2165), pHER2 Y877 (Abcam, rabbit mAb #2241), pHER2 Y1248 (Abcam, rabbit mAb #2247), pHER2 Y1222 (Abcam, rabbit mAb #2243), pHER2 Y1196 (Abcam, rabbit mAb #6942), pHER2 Y1112 (Millipore, mouse mAb #04-294), αV-integrin (Abcam, rabbit mAb #ab179475), β6-integrin (Santa Cruz Biotechnology, goat pAb #sc-6632), β1-integrin (Abcam, rabbit mAb #ab52971), vinculin (Abcam, mouse mAb #ab11194), paxillin (BD, mouse mAb #610051), phospho-Erk 1/2 (p44/42 MAPK) T202/Y204 (pERK1/2) (Cell Signaling Technology, rabbit mAb #137F5), Akt (Cell Signaling Technology, rabbit mAb #4691), pAkt (Cell Signaling Technology, rabbit mAb #4060), GAPDH (Abcam, mouse mAb #ab9484), or β-actin (Sigma-Aldrich, mouse mAb #A3853) were incubated at 4°C overnight.

Techniques: Activity Assay, Membrane, Migration, Activation Assay, Clinical Proteomics

Journal: Neurobiology of disease

Article Title: Overexpression of alpha synuclein disrupts APP and Endolysosomal axonal trafficking in a mouse model of synucleinopathy

doi: 10.1016/j.nbd.2023.106010

Figure Lengend Snippet: The overexpression of GFP-ASYN induced retrograde axonal transport deficits of Rab5 and Rab7 in E18 cortical neurons. E18 cortical neurons from Line 78 (PDGF-β-ASYN-GFP) and SynKO mouse embryos were dissected and cultured in microfluidic chambers as described in . At DIV14, neurons were transfected with mCherry-WT-Rab5 (A-E) and mCherry-WT-Rab7 (F-J). After 24hs, axonal transport of Rab5 and Rab7 was captured by live imaging. Kymographs were generated from time-lapsed image series. Representative image of Rab5 and Rab7 within axons of non-transgenic (A, F), ASYN-knockout (B, G) and PDNG78 transgenic neurons (C, H) are shown. Axonal transport parameters: the retrograde moving velocity (D, I) and average velocity (E, J) are quantitated and presented. Data were obtained from 20 non-transgenic neurons, 20 ASYN-knockout neurons and 15 GFP-positive transgenic neurons. Representative images of axons correspond to the first frame of the kymograph. All data are analyzed using Prism GraphPad 6.0. The p values were obtained using student t-test. p < 0.05 (*); p < 0.001 (***); n.s. = non-significant.

Article Snippet: Rab5 and Rab7 were stained with anti-mouse Rab5 rabbit primary antibody (abcam EPR21801 and EPR7589), followed by anti rabbit AF-594-tagged secondary antibody (Invitrogen).

Techniques: Over Expression, Cell Culture, Transfection, Imaging, Generated, Transgenic Assay, Knock-Out

Journal: Neurobiology of disease

Article Title: Overexpression of alpha synuclein disrupts APP and Endolysosomal axonal trafficking in a mouse model of synucleinopathy

doi: 10.1016/j.nbd.2023.106010

Figure Lengend Snippet: Colocalization of GFP-hASYN with Rab5 in E18 PDNG78 cortical neurons. E18 cortical neurons were cultured and fixed for immunostaining with specific Abs against Rab5 (red), Rab7, Lamp1. (A) Representative images for GFP-hASYN (green), Rab5 (red), Rab7 (red), and Lamp1 (red) are shown. All nuclear staining (DAPI) is shown in blue. (B) Insets were enlarged to better visualize the colocalization between hASYN and these three different markers. (C) Colocalization was quantified as Pearson’s correlation coefficients for at least three cells for each experiment.

Article Snippet: Rab5 and Rab7 were stained with anti-mouse Rab5 rabbit primary antibody (abcam EPR21801 and EPR7589), followed by anti rabbit AF-594-tagged secondary antibody (Invitrogen).

Techniques: Cell Culture, Immunostaining, Staining

Journal: bioRxiv

Article Title: Endogenous 1-deoxysphingolipid synthesis compromises anchorage-independent growth and plasma membrane endocytosis in cancer cells

doi: 10.1101/2022.01.19.476986

Figure Lengend Snippet: A. RAB5 immunostaining with SPTLC1 WT and SPTLC1 C133W expressing HCT116 cultured for 4 days with 0.1 µg/ml doxycycline. Dynasore (dyn) was given with 20 µM for 3h. B. RAB5 immunostaining with SPTLC1 WT and SPTLC1 C133W expressing A549 cultured for 4 days with 1 µg/ml doxycycline. Dynasore (dyn) was given with 20 µM for 3h. C. RAB5 immunostaining with HCT116 cells cultured with 1mM alanine or 1mM serine. Violin blots depict relative RAB5 intensity obtained from n= cell number. Data are representative of two independent experiments. White scale bar indicates 10 µm.

Article Snippet: Cells were then incubated with primary anti-RAB5 antibody at 4 °C overnight diluted in 8% BSA in PBST (1:500 dilution, Cat. #3547, lot 7, Cell Signaling Technology).

Techniques: Immunostaining, Expressing, Cell Culture