Rab10

Manufactured by Cell Signaling Technology

Rab10 is a small GTPase protein that is involved in intracellular membrane trafficking processes. It plays a role in the regulation of vesicle formation, transport, and fusion within the cell. Rab10 is a member of the Rab family of proteins, which are known to be important regulators of various cellular functions.

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10 protocols using rab10

Western Blot Analysis of Cellular Proteins

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Proteins were extracted in RIPA buffer and quantified using the Pierce BCA kit (Thermo Fisher). Totally, 20–30 µg of protein was boiled in NuPage buffer (Thermo Fisher) and loaded onto a denaturing SDS-polyacrylamide gel (10%), transferred to PVDF membranes and blotted with anti-mouse or -rabbit HRP-conjugated secondary antibodies (Bio-Rad). The following antibodies were used for immunoblotting: Gal-3 (Cell Signaling), RAB10 (Cell Signaling), β1 integrin (P4C10, Millipore), pAKT (Cell Signaling), AKT (Cell Signaling), pERK (Cell Signaling), ERK (Cell Signaling), and β-actin HRP (Sigma-Aldrich) as loading control. For protein expression analysis, expression was normalized to β-actin then compare to their respective control. For analysis of AKT and ERK activation, p-Akt and p-ERK were normalized to total AKT and total ERK, respectively, then compared to their control.

Seguin L., Odouard S., Corlazzoli F., Haddad S.A., Moindrot L., Calvo Tardón M., Yebra M., Koval A., Marinari E., Bes V., Guérin A., Allard M., Ilmjärv S., Katanaev V.L., Walker P.R., Krause K.H., Dutoit V., Sarkaria J.N., Dietrich P.Y, & Cosset É. (2021). Macropinocytosis requires Gal-3 in a subset of patient-derived glioblastoma stem cells. Communications Biology, 4, 718.

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Insulin Signaling Pathway Protein Analysis

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Primary antibodies used: Akt #9272 (1:1000), p-Akt (Ser473) # 4058 (1:1000), p-Akt (Thr308) #2965, Rab8A #6975 (1:1000), Rab10 #8127 (1:1000), AS160 #2670 (1:1000), p-AS160 (Thr642) #4288 (1:1000), IR beta #3025 (1:1000), p-IGF-IR beta (Tyr1131)/IR beta (Tyr1146) #3021 (1:1000) (all from Cell Signalling); LRRK2 (3514-1 (MJFF2) Epitomics); tubulin (ab6160, abcam); SLC2A4 (ARP43785_P050, avivasysbio, 1:100); GLUT4 (MAB1262, R&D Systems, 1:1000); Alexa Fluor 488 Mouse anti-β-Tubulin, Class III Clone TUJ1 #560338 (BD Pharmingen).
Secondary antibodies used: anti-rabbit IgG, HRP-linked #7074 (1:5000, Cell signalling); Goat anti-rat IgG, HRP-linked (1:20 000, ab6845, abcam); Rabbit anti-sheep IgG, HRP-linked (1:10 000, 31480, ThermoScientific); Goat anti-mouse IgG, HRP-linked (1:10 000, 172–1011, Bio-Rad); Cy3-conjugated Goat anti-rabbit IgG (1:350, 111-165-046, Jackson ImmunoResearch); Cy2-conjugated Goat anti-mouse IgG (1:350, 115-225-164, Jackson ImmunoResearch).

Funk N., Munz M., Ott T., Brockmann K., Wenninger-Weinzierl A., Kühn R., Vogt-Weisenhorn D., Giesert F., Wurst W., Gasser T, & Biskup S. (2019). The Parkinson’s disease-linked Leucine-rich repeat kinase 2 (LRRK2) is required for insulin-stimulated translocation of GLUT4. Scientific Reports, 9, 4515.

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Western Blot Analysis of Cellular Proteins

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Total cell protein extracts were prepared as mentioned above and 50 µg of protein was used for western blotting performed according to standard protocols. Antibodies used were PER21A from Alpha Diagnostics International, RAB1 from Sigma-Aldrich, SARA1; GAPDH and RAB10 from Cell Signaling. The density of the bands obtained in the western blots was calculated with the freely available gel analyzer program ImageJ.

Robles M.S., Cox J, & Mann M. (2014). In-Vivo Quantitative Proteomics Reveals a Key Contribution of Post-Transcriptional Mechanisms to the Circadian Regulation of Liver Metabolism. PLoS Genetics, 10(1), e1004047.

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Immunoblotting Analysis of Insulin Signaling Proteins

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Samples were run on 4–20% precast SDS–PAGE gels (Novex, Life Technologies) and transferred to nitrocellulose membrane. Antibodies to HA and Myc (monoclonal mouse and polyclonal rabbit) and human Rlf were purchased from Santa Cruz Biotechnology. Mouse-FLAG antibodies were from Agilent Technologies. Anti-RFP antibodies were from Abcam (Cambridge, MA). AS160 and pAS160 (T642) antibodies were from EMD Millipore. Rabbit anti-Rlf serum described previously (Wolthuis et al., 1997 (link)) was affinity purified (Pacific Immunologies, Ramona, CA) and used for immunoblotting. RalA and caveolin3 antibodies were from BD Biosciences (San Jose, CA). Transferrin receptor antibody was from Zymed Labs. Glut4 and Sec8 antibodies were purchased from Alpha Diagnostics (San Antonio, TX) and Enzo LifeSciences (Farmingdale, NY). Rab10, Akt, pAkt (S473), pAkt-substrate, histone H3, and IRAP antibodies were from Cell Signaling (Danvers, MA). Horseradish peroxidase–conjugated secondary antibodies were obtained from Thermo Scientific (Waltham, MA), and Alexa Flour secondary antibodies were from Molecular Probes, Life Technologies. Insulin, GDP, GTPγS, and Optiprep gradient solution were purchased from Sigma-Aldrich (St. Louis, MO).

Karunanithi S., Xiong T., Uhm M., Leto D., Sun J., Chen X.W, & Saltiel A.R. (2014). A Rab10:RalA G protein cascade regulates insulin-stimulated glucose uptake in adipocytes. Molecular Biology of the Cell, 25(19), 3059-3069.

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AMPK Signaling Pathway Profiling

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Unless otherwise stated all reagents were from Sigma-Aldrich. Primary antibodies against pan-AMPK-α (#2532), pAMPK Thr¹⁷² (#2531), ACC (#3876), pACC Ser⁷⁹ (#3661), EGFR (#2232), IRAP (#3808), N-cadherin (#14215), Rab10 (#8127S) and rabbit FLAG (#2368) were from Cell Signaling Technology. CD36 (ab124515), Calmodulin (ab45689), GLUT1 (ab115730) and SERCA1 (ab129104) were from Abcam. From Santa Cruz Biotechnology Inc were antibodies against pan-14-3-3 (sc-629) and transferrin receptor (sc-65882). Other antibodies were EHBP1L1 (#PAB15793; Abnova), GFP (118144600001; Roche), HA.11 16B12 (901503; Biolegend), MICAL1 (14818–1-AP; proteintech), pTBC1D1 Ser²³⁷ (#07-2268; Merck-Millipore), VPS13C (HPA043356; Atlas Antibody), β actin (A1978), Tubulin (T9026) and mouse FLAG (F1804). VPS13A antibody was produced as previously described^{84 (link)}. Secondary antibodies used were conjugated with an AlexaFluor (Invitrogen) or HRP (Cell Signaling Technology or Jackson ImmunoResearch). AICAR was from Tocris.

Hook S.C., Chadt A., Heesom K.J., Kishida S., Al-Hasani H., Tavaré J.M, & Thomas E.C. (2020). TBC1D1 interacting proteins, VPS13A and VPS13C, regulate GLUT4 homeostasis in C2C12 myotubes. Scientific Reports, 10, 17953.

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Protein Analysis in Neurodegenerative Disorders

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Primary antibodies for western blotting were VPS13C (HPA043507, 1:500; Sigma-Aldrich, 28676-1-AP, 1:800; Proteintech), phosphoRab10-T73 (230261, 1:500; Abcam), Rab10 (8127, 1:1,000; Cell Signaling), βIII-Tubulin (4466S, 1:4,000; Biolegend), TH (657012, 1:2,000; Millipore), GAPDH (2,118, 1:2,000; Millipore), α-Tubulin (5168, 1:20,000; Sigma-Aldrich), LAMP2 (H4B4, 1:1,000; DSHB), GCase (G4171, 1:1,000; Sigma-Aldrich), Rab7 (137029, 1:1,000; Abcam), HA (3724, 1:2,000; Cell Signaling), Calnexin (1:1,000; Cell Signaling), TOM20 (612278, 1:1,000; BD Biosciences), PEX5 (83020S, 1:500, Cell Signaling), GFP (1544, 1:2,000; Sigma-Aldrich), Myc (2278, 1:2,000; Cell Signaling), cathepsin B (AF953, 1:2,000; R&D systems), cathepsin D (6487, 1:1,000; Santa Cruz), LRRK2 (ab133474, 1:500; Abcam), LRRK2-S935 (ab133450, 1:500; Abcam), EHBP1 (17637-1-AP, 1:1,000; Proteintech), and PPM1H (PA5-26102, 1:1,000; Invitrogen).
Primary antibodies for immunofluorescence were βIII-Tubulin (4466S, 1:300; Biolegends), TH (657012, 1:300; Millipore), and LAMP1 (sc-20011, 1:300; Santa Cruz).
Primary antibodies for PLA were VPS13C (28676-1-AP, 1:100; Proteintech) and Rab10 (ab104859, 1:100; Abcam).

Schrӧder L.F., Peng W., Gao G., Wong Y.C., Schwake M, & Krainc D. (2024). VPS13C regulates phospho-Rab10-mediated lysosomal function in human dopaminergic neurons. The Journal of Cell Biology, 223(5), e202304042.

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Comprehensive Antibody Panel for Neuronal Characterization

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Rab10 (4262S, Cell Signaling Technology) for immunoblot, Rab10 (ab237703, Abcam) for immunofluorescence, pRab10 for immunoblot (ab230261, Abcam), pRab10 for immunofluorescence (ab241060, Abcam), Hsc70 (ab19136, Abcam), α-Tubulin (SC23948, Santa Cruz Biotechnology), parvalbumin, (NBP2-50036, NovusBio), NeuN (MAB377, Millipore), SATB2 (ab51502 Abcam), calretinin (MAB1568, Millipore), DARPP32 (MAB4230, R&D Systems), ChAT (NBP2-46620, NovusBio), DAT loop (6-8D6 Santa Cruz Biotechnology), tyrosine hydroxylase (TH) (ab76442, Abcam), CD68 (NBP2-33337SS, NovusBio), GFAP (AB5541, Millipore), Olig2 (MABN50 Millipore), KDEL receptor (sc-58774, Santa Cruz Biotechnology), TGN46 (MA3-063, ThermoFisher), LAMP1 (1D4B, DHSB), EEA1 (NBP2-36568, NovusBio), α-Synuclein (610786, BD Transduction Lab), Synuclein (ab51252, Abcam), VAMP2 (104 211, Synaptic Systems), Homer1 (160 006 Synaptic Systems), Rab8a (ab188574).

Singh V., Menard M.A., Serrano G.E., Beach T.G., Zhao H.T., Riley-DiPaolo A., Subrahmanian N., LaVoie M.J, & Volpicelli-Daley L.A. (2023). Cellular and subcellular localization of Rab10 and phospho-T73 Rab10 in the mouse and human brain. Acta Neuropathologica Communications, 11, 201.

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Western Blot Analysis of Insulin Signaling

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Tissues were processed using a multisample pulverizer and were prepared in lysis buffer (catalog #9803S; Cell Signaling Technology) containing HALT inhibitor cocktail (catalog #78442; Thermo Scientific). An equivalent amount of protein was used for all samples in each gel. Membranes were incubated with primary antibodies at 4°C overnight, as follows: Rab10 (1:500; catalog #4262; Cell Signaling Technology), GLUT4 (1:25,000; catalog #2213; Cell Signaling Technology), phosphorylated AS160 T642 (1:1,000; catalog #8881; Cell Signaling Technology), AS160 (1:5,000; catalog #07–741; Millipore), Rab8a (1:1,000; catalog #610844; BD Biosciences), phosphorylated Akt S473 (1:2,000; catalog #9271; Cell Signaling Technology), and Akt (1:1,000; catalog #9272; Cell Signaling Technology).

Vazirani R.P., Verma A., Sadacca L.A., Buckman M.S., Picatoste B., Beg M., Torsitano C., Bruno J.H., Patel R.T., Simonyte K., Camporez J.P., Moreira G., Falcone D.J., Accili D., Elemento O., Shulman G.I., Kahn B.B, & McGraw T.E. (2016). Disruption of Adipose Rab10-Dependent Insulin Signaling Causes Hepatic Insulin Resistance. Diabetes, 65(6), 1577-1589.

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Western Blot Analysis of 3T3-L1 Adipocytes

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Western blot experiments were performed using standard protocols. Cellular protein extracts were collected from 3T3-L1 differentiated adipocytes expressing indicated siRNA or DNA constructs. Antibodies used were as follows: RAB10 (4262S; Cell Signaling Technology); AS160 (07–741; EMD Millipore); ACTIN (AAN01-A; Cytoskeleton, Inc.); AKT (9272S; Cell Signaling Technology); P-AKT (9271S; Cell Signaling Technology); P-AS160 (8881S; Cell Signaling Technology); TR (ab84036; Abcam); IRAP (6918P; Cell Signaling Technology); GFP (Roche); and SEC16A (MKA0310; Cosmo Bio Co.).

Bruno J., Brumfield A., Chaudhary N., Iaea D, & McGraw T.E. (2016). SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes. The Journal of Cell Biology, 214(1), 61-76.

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Protein Expression Analysis of Neutrophils

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After 24 h, neutrophils treated with gradient glutamate were lysed in 1× RIPA lysis buffer (P0013C; Beyotime) and Protease Inhibitor Cocktail (78429; Thermo Fisher Scientific). Antibodies of ARF4 (bs‐8750R; Bioss), RAB10 (8127; Cell Signaling Technology), pSTAT3 (Tyr705, 9145; Cell Signaling Technology), and β‐actin (3700S; Cell Signaling Technology) were incubated overnight. Goat anti‐rabbit/mouse HRP tagged secondary antibody (A0208 and A0216; Beyotime) were subsequently incubated at room temperature for 1 h. Finally, protein blots were visualized western ECL substrate (1705060; Bio‐Rad).

Xiong T., He P., Zhou M., Zhong D., Yang T., He W., Xu Z., Chen Z., Liu Y, & Dai S. (2022). Glutamate blunts cell‐killing effects of neutrophils in tumor microenvironment. Cancer Science, 113(6), 1955-1967.

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