Mouse anti rab5

Manufactured by BD

Sourced in United States, Germany

Mouse anti-Rab5 is a primary antibody that recognizes the Rab5 protein, a small GTPase involved in early endosome formation and trafficking.

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Anti-Rab5 (7 citations) Mouse anti (2 citations) Mouse monoclonal anti-Rab5 (2 citations)

7 protocols using mouse anti rab5

Immunoblotting and Microscopy Analysis

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Mouse anti‐Tetherin (APC coupled, BioLegend, 348410) and mouse anti‐CD4 (PE coupled, BioLegend, 300508) were used for flow‐cytometry and FACS. Antibodies used for immunoblotting include: rabbit anti‐WDR81 (Abcam, ab1217333, LOT GR98370‐3); rabbit anti‐Tetherin [NIH AIDS Reagent Programme, 11721, LOT 110114, 83]; rabbit anti‐Phospho‐EGF Receptor (Cell Signalling, 2234); mouse anti‐MHC‐I heavy chain (clone 3B10.7); mouse anti‐Fatty Acid Synthase (BD, 610962); mouse anti‐α‐tubulin (Sigma, T9026); mouse anti‐ß‐actin (Sigma, A2228); mouse anti‐calnexin (BD, 610962); goat anti‐mouse‐HRP (Jackson ImmunoResearch, 115‐035‐062) and goat anti‐rabbit‐HRP (Jackson ImmunoResearch, 111‐035‐144). Antibodies used for confocal microscopy: rabbit anti‐Tetherin [NIH AIDS Reagent Programme, 11721, LOT 110114, 83]; mouse anti‐HA (BioLegend, 901502); mouse anti‐EEA1 (BD, 610457); mouse anti‐Rab5 (BD, 610724); mouse anti‐CD63 (BD, 556019); mouse anti‐LAMP1 (BD, 555798); mouse anti‐LAMP1 (Alexa Fluor 647 conjugated, BD, 562622); rabbit anti‐Cathepsin D (Millipore, 219361); donkey anti‐rabbit IgG (Alexa Fluor 488 conjugate, Thermo Fisher Scientific, A‐21206); donkey anti‐mouse IgG (Alexa Fluor 647 conjugate, Thermo Fisher Scientific, A‐31571); goat anti‐mouse IgG (Pacific Blue conjugate, Thermo Fisher Scientific, P31582).

Rapiteanu R., Davis L.J., Williamson J.C., Timms R.T., Paul Luzio J, & Lehner P.J. (2016). A Genetic Screen Identifies a Critical Role for the WDR81‐WDR91 Complex in the Trafficking and Degradation of Tetherin. Traffic (Copenhagen, Denmark), 17(8), 940-958.

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Cytoskeleton Disruption and Protein Synthesis Inhibition in HUVECs

Cited 2 times

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Latrunculin B (Calbiochem-Novabiochem Corp., San Diego, CA, USA) and nocodazole (Sigma-Aldrich, Saint Louis, MO, USA) were used as previously described to disrupt actin and microtubule cytoskeleton integrity, respectively [27 (link)]. Cycloheximide (Sigma-Aldrich) was used to inhibit protein synthesis in HUVECs at the concentration of 50 μg/mL.
The following primary antibodies were used: mouse monoclonal anti-CD93 (mAb 4E1) [6 (link)], rabbit anti-MMRN2 (generously provided by M. Mongiat), rabbit anti-CD93 (HPA009300, Atlas Antibodies, Bromma, Sweden), mouse anti-CD93 (MBL International Corporation, Woburn, MA, USA), rabbit anti-Giantin, mouse anti-β1 integrin (12G10), and mouse anti-Rab7 (Abcam, Cambridge, UK), rabbit anti-Moesin (Cell Signaling Technology, Danvers, MA, USA), mouse anti-Rab5 (BD Biosciences, Franklin Lakes, NJ, USA), mouse anti-β-actin (Sigma-Aldrich), rabbit anti-CD93 (H-190), mouse anti-COPD (E-12), mouse anti-Sec31A (H-2), mouse anti-β-Adaptin (A-5), mouse anti-Rab5a (E-11), mouse anti-Rab5b (F-9), mouse anti-Rab5c (H-3), mouse anti-β1 integrin (4B7R), mouse anti-Rab11a (D-3), rabbit anti-caveolin-1 (N-20), and mouse anti-MMRN2 (H572) (Santa Cruz Biotechnology, Dallas, TX, USA). Alexa Fluor-488 and -647 phalloidin (Thermo Fisher Scientic) were used for F-actin labeling.

Barbera S., Nardi F., Elia I., Realini G., Lugano R., Santucci A., Tosi G.M., Dimberg A., Galvagni F, & Orlandini M. (2019). The small GTPase Rab5c is a key regulator of trafficking of the CD93/Multimerin-2/β1 integrin complex in endothelial cell adhesion and migration. Cell Communication and Signaling : CCS, 17, 55.

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Antibody Panel for EGFR Signaling Analysis

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Mouse anti-HA (MMS-101P, Biolegend, San Diego, CA), mouse anti-phospho tyrosine (PY72) (P172.1, InVivo Biotech Services, Henningsdorf, Germany), rabbit anti-c-Cbl (sc-170, Santa Cruz Biotechnologies, Heidelberg, Germany), mouse monoclonal anti-α-Tubulin (Sigma Aldrich, St. Louis, MO); living colors rabbit anti-GFP (632593, Clontech, Mountain View, CA), living colors mouse anti-GFP (632681, Clontech, Mountain View, CA), mouse anti-Rab11 (610656, BD Biosciences, Heidelberg, Germany), rabbit anti-Rab11a (ab65200, Abcam), rabbit anti-Rab11 (3539, Cell Signaling Technology, Danvers, MA), rabbit EGFR (4267, Cell Signaling Technology, Danvers, MA), rabbit pY1045 (2237, Cell Signaling Technology, Danvers, MA), rabbit pY1068 (3777, Cell Signaling Technology, Danvers, MA), goat EGFR (AF231, R&D Systems, Minneapolis, MN), mouse pY845 (558381, BD Biosciences, Heidelberg, Germany), mouse anti-Rab5 (610281, BD Biosciences, Heidelberg, Germany), rabbit anti-phospho ERK-1/2 Thr/Tyr 202/204 (9101, Cell Signaling Technology, Danvers, MA), mouse anti-ERK1/2 (Ab366991, Abcam); rabbit anti phosphor-Akt Ser473 (9271, Cell Signaling Technology, Danvers, MA), mouse anti-Akt (pan) (2920, Cell Signaling Technology, Danvers, MA).

Baumdick M., Brüggemann Y., Schmick M., Xouri G., Sabet O., Davis L., Chin J.W, & Bastiaens P.I. (2015). EGF-dependent re-routing of vesicular recycling switches spontaneous phosphorylation suppression to EGFR signaling. eLife, 4, e12223.

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Western Blot Analysis of Rab GTPases

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Treated cells were collected in Laemmli sample buffer, sonicated and boiled. Samples were run on NuPage 4%–12% Bis-Tris gel (Life Technologies), transferred to PVDF membranes (Perkin Elmer), blocked in 5% skimmed milk and incubated successively with primary and secondary-HRP coupled antibodies, and finally visualized with ECL (Thermo Scientific) or Luminata Crescendo (Millipore) HRP reagents depending on the strength of the signals. Signals were captured on Amersham Hyperfilm ECL (GE Healthcare), developed using a Xograph Compact X4 film developer and analyzed using ImageJ software (National Institutes of Health, USA). Signals used for quantifications were captured at a pre-saturation intensity. Results are derived from triplicate biological repeats and represent signals that were normalized to a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading control and to the signals from resting cells. Antibodies used were mouse anti-Rab4 (BD Biosciences 610888), mouse anti-Rab5 (BD Biosciences 610282), rabbit anti-Rab7 (Cell Signaling Technologies D95F2), rabbit anti-Rab8 (Cell Signaling Technologies D22D8), rabbit anti-Rab11 (Life Technologies, 715300), mouse anti-GAPDH (Santa Cruz 0411), and rabbit anti-GAPDH (Cell Signaling Technologies 14C10).

Augustin H., McGourty K., Allen M.J., Adcott J., Wong C.T., Boucrot E, & Partridge L. (2018). Impact of insulin signaling and proteasomal activity on physiological output of a neuronal circuit in aging Drosophila melanogaster. Neurobiology of Aging, 66, 149-157.

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Neuronal Immunostaining Protocol

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After treatments, neurons were washed twice with 37°C 4% sucrose/1X phosphate-buffered-saline (PBS; 10X: 1.4 M NaCl, 26.8 mM KCl, 62 mM Na₂HPO₄, 35.3 mM KH₂PO₄, pH 7.4), then fixed for 15 min with 4% sucrose/4% formaldehyde (Thermo Fisher Scientific) in 1X PBS. Neurons were washed 3 × 5 min with 1X PBS, permeabilized for 10 min with 0.2% Triton X-100 (Amresco, Solon, OH) in 1X PBS, and blocked for 30 min in 5% normal donkey serum (Jackson ImmunoResearch, West Grove, PA) in 1X PBS. Neurons were then incubated in primary antibody diluted in block for 1 h at RT, washed 3 × 5 min in 1X PBS, and incubated in secondary antibody diluted in block for 1 h at RT. Neurons on coverslips were mounted on glass slides in Fluoromount (Thermo Fisher Scientific) and dried overnight at RT. Primary antibodies used were: rabbit anti-Arc (1:1000; custom-made; ProteinTech, Rosemont, IL); rabbit anti-Arc (1:1000; Synaptic Systems, Goettingen, Germany); chicken anti-MAP2 (1:5000; ab5392; Abcam); mouse anti-Rab5 (1:1000; BD Biosciences, San Jose, CA); DAPI nuclear stain (Molecular Probes, Thermo Fisher Scientific). Secondary antibodies used were: Alexa Fluor 405, 488, 555, or 647 for the appropriate animal host (1:750; Thermo Fisher Scientific or Jackson ImmunoResearch).

Pastuzyn E.D., Day C.E., Kearns R.B., Kyrke-Smith M., Taibi A.V., McCormick J., Yoder N., Belnap D., Erlendsson S., Morado D.R., Briggs J.A., Feschotte C, & Shepherd J.D. (2018). The Neuronal Gene Arc Encodes a Repurposed Retrotransposon Gag Protein that Mediates Intercellular RNA Transfer. Cell, 172(1-2), 275-288.e18.

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Immunostaining Protocol for iMNs

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iMNs were fixed in 4% paraformaldehyde (PFA) for 1h at 4 ºC, permeabilized with 0.5% PBS-T overnight at 4 ºC, blocked with 10% FBS in 0.1% PBS-T at room temperature for 2 h, and incubated with primary antibodies at 4 ºC overnight. Cells were then washed with 0.1% PBS-T and incubated with Alexa Fluor® secondary antibodies (Life Technologies) in blocking buffer for 2 h at room temperature. To visualize nuclei, cells were stained with DAPI (Life Technologies) then mounted on slides with Vectashield® (Vector Labs). Images were acquired on an LSM 780 confocal microcope (Zeiss). The following primary antibodies were used: mouse anti-HB9 (Developmental Studies Hybridoma Bank); mouse anti-TUJ1 (EMD Millipore); rabbit anti-VACHT (Sigma); rabbit anti-C9ORF72 (Sigma-Aldrich); mouse anti-EEA1 (BD Biosciences); mouse anti-RAB5 (BD Biosciences); mouse anti-RAB7 (GeneTex); mouse anti-LAMP1 (Abcam); mouse anti-LAMP3 (DSHB, cat. no. H5C6); rabbit anti-LAMP3 (Proteintech, cat. no. 12632); mouse anti-LAMP2 (DSHB, cat. no. H4B4); mouse anti-M6PR (Abcam, cat. no. Ab2733); rabbit anti-GluR1 (EMD Millipore, cat. no. pc246); mouse anti-GluR1 (Santa Cruz); rabbit anti-NR1 (EMD Millipore); mouse anti-NR1 (EMD Millipore, cat. no. MAB363); chicken anti-GFP (GeneTex).

Shi Y., Lin S., Staats K.A., Li Y., Chang W.H., Hung S.T., Hendricks E., Linares G.R., Wang Y., Son E.Y., Wen X., Kisler K., Wilkinson B., Menendez L., Sugawara T., Woolwine P., Huang M., Cowan M.J., Ge B., Koutsodendris N., Sandor K.P., Komberg J., Vangoor V.R., Senthilkumar K., Hennes V., Seah C., Nelson A.R., Cheng T.Y., Lee S.J., August P.R., Chen J.A., Wisniewski N., Victor H.S., Belgard T.G., Zhang A., Coba M., Grunseich C., Ward M.E., van den Berg L.H., Pasterkamp R.J., Trotti D., Zlokovic B.V, & Ichida J.K. (2018). Haploinsufficiency Leads to Neurodegeneration in C9ORF72 ALS/FTD Human Induced Motor Neurons. Nature medicine, 24(3), 313-325.

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Antibodies for ESCRT-II and Vesicular Trafficking

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Rabbit anti-Xenopus laevis ESCRT-II polyclonal antibody was made in the Blower laboratory; other antibodies were obtained as follows: mouse anti-acetylated α-tubulin (Sigma cat. no. T6793), mouse anti-α-tubulin (Sigma cat. no. T6074), mouse anti-Rab4 (BD Biosciences cat. no. 610888), mouse anti-Rab5 (BD Biosciences cat. no. 610281), mouse anti-Rab7 (Sigma cat. no. R8779), mouse anti-Rab11 (BD Biosciences cat. no. 610656), rabbit anti-β-tubulin (Abcam cat. no. ab6046), mouse anti-DCC intracellular domain (BD Biosciences cat. no. 554223), goat anti-DCC extracellular domain (R&D Systems cat. no. AF844), rabbit anti-RPL5 (Proteintech Europe cat. no. 15430-1-AP).

Konopacki F.A., Wong H.H., Dwivedy A., Bellon A., Blower M.D, & Holt C.E. (2016). ESCRT-II controls retinal axon growth by regulating DCC receptor levels and local protein synthesis. Open Biology, 6(4), 150218.

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