Protein samples were collected from cells or exosomes in RIPA lysis buffer (Solarbio, Beijing, China). The processed protein samples were separated by 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.22 μm PVDF membranes (Millipore), and incubated with primary antibodies against CD63, TSG101, Rab11a, and GAPDH (Abcam, Cambridge, MA, United States) at 4 °C. The secondary antibody was goat anti-rabbit antibody (Abcam, Cambridge, MA, United States) and incubation was performed at 24 °C. Chemiluminescence reagent (Millipore) was used to detect the proteins.
Rab11a
Manufactured by Abcam
Sourced in United Kingdom
Rab11a is a small GTPase protein that is involved in the regulation of endocytic recycling pathways. It plays a key role in the trafficking and recycling of various proteins and membrane components within the cell.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescence reagent, by Merck Group (1 mentions)
0.22 μm pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Integrin antibody sampler kit, by Cell Signaling Technology (1 mentions)
Pi3k c2α, by Abcam (1 mentions)
Kif15, by Abcam (1 mentions)
Goat anti rabbit secondary antibody, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Stem cell factor (scf), by R&D Systems (1 mentions)
Eht1864, by Bio-Techne (1 mentions)
Sdf 1α, by R&D Systems (1 mentions)
12 o tetradecanoylphorbol 13 acetate tpa, by Merck Group (1 mentions)
Mbq 167, by Selleck Chemicals (1 mentions)
Nexinhib20, by Bio-Techne (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Gm6001, by Merck Group (1 mentions)
Caveolin 1, by Cell Signaling Technology (1 mentions)
Fluorescently labeled phalloidin, by Thermo Fisher Scientific (1 mentions)
Myosin vb, by Novus Biologicals (1 mentions)
7 protocols using rab11a
1
Western Blot Analysis of Exosomal Proteins
2
Western Blotting of Cell Signaling Proteins
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Western blotting was performed as previously described [17 (link)]. Primary antibodies against KIF15, FAK, FAK-397, PXN, PXN-118, SRC, SRC-418, RAB11A, PI3K-C2α, and FLAG were purchased from Abcam. The Integrin Antibody Sampler Kit and antibody against GAPDH were purchased from Cell Signaling Technology. HRP-conjugated secondary antibodies, including polyclonal goat mouse IgG and polyclonal goat rabbit IgG, were purchased from Cell Signaling. GADPH was used as a normalization loading control. Expression levels were quantified by Imaging Systems (Thermo Fisher), and the relative expression values were quantified using Image J software.
3
Immunoblotting analysis of RAB11A and GAPDH
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Before protein extraction, TC cells were washed with 1*PBS to remove the culture media. Cells were collected and lysed with RIPA lysis buffer at 4°C, and lysates were centrifuged at 12,000 rpm for five minutes at 4°C. Forty micrograms of cellular proteins were separated by 12% SDS-PAGE, and were transferred to PVDF membrane (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk at room temperature for two hours, the membranes were probed with primary antibodies at 4°C overnight. Then, the membranes were incubated with corresponding secondary antibodies at room temperature for two hours. The primary antibodies used in this study included RAB11A (Abcam, Cambridge, UK) and GAPDH (Abcam, Cambridge, UK), and the secondary goat anti-rabbit antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA).
4
Evaluation of Stem Cell Signaling Modulators
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Stem cell factor (SCF), stromal cell-derived factor 1 alpha (SDF-1α) and interleukin-3 (IL-3) were obtained from R&D systems (Minneapolis, MN). Chemical inhibitors PP3, EHT 1864, and Nexinhib20 were obtained from Tocris (Minneapolis, MN). Ascorbic acid and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were obtained from Sigma-Aldrich (St. Louis, MO). MBQ-167 was obtained from Selleckchem (Houston, TX). Recombinant fibroblast growth factor 2 (FGF-2), antibody against β-Actin and the chemical inhibitors PP2 and GM6001 were obtained from EMD Millipore (Billerica, MA). Antibodies against Rab3B, Rab3D, Rab11A and Rab27A were obtained from Abcam (Cambridge, MA). Antibodies against Caveolin-1, Rab3A, Rab5 and Rab8 were obtained from Cell Signaling Technology (Danvers, MA). An antibody against RalA was obtained from BD Biosciences (San Jose, CA). GFP-RalA and S-Ch-Cdc42 adenoviruses were generated and utilized as described previously[13 (link), 40 (link)].
5
Quantification of Mechanosensitive Proteins
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Equivalent amounts of cell lysate (10 μg) extracted from cells cultured under static or OSS conditions for 96h were western blotted with the following antibodies: Dab2 (D709T, Cell Signaling Technology #12906); Caveolin-1 (Cell Signaling Technology #3238); Rab11a (Abcam #ab65200); and Dynamin-2 (Hudy-1, EMD Millipore #MABT188). Band intensities were quantified using and background subtracted. Intensities of OSS-exposed samples were normalized to control (Static) values (set at 100%) and significance was assessed separately for each antibody in GraphPad Prism software using a one sample t-test.
6
Immunoblotting Antibodies Catalogue Reference
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Antibodies used in this study were ABCC2/ MRP2 (catalogue number MAB4150; Millipore, Burlington, MA), RAB11A (mouse) (catalogue number 610656; BioScience, Franklin Lakes, NJ), RAB11A (rabbit) (catalogue number ab128913; Abcam, Cambridge, MA), myosin Vb (catalogue number NBP1-87746; Novus), FLAG (catalogue number F3165; Sigma-Aldrich, St Louis, MO), UNC45A (rabbit) (catalogue number HPA039228; Merck, Kenilworth, NJ), UNC45A (mouse) (catalogue number ADI-SRA-1800-F; Enzo Life Sciences, Farmington, NY), beta-tubulin (catalogue number T4026; Merck), c-myc (catalogue number 631206; Takara, Kusatsu, Japan), ezrin (catalogue number SC58758; Santa Cruz Biotechnology, Dallas, TX), beta-actin (catalogue number A5441; Sigma-Aldrich), syntaxin-3 (catalogue number ab133750; Abcam), and munc18-2 (catalogue number ab103976; Abcam). Fluorescently labeled phalloidin (catalogue number A22284) was from Invitrogen.
7
Immunoblotting of Rab GTPases
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Protein samples were extracted from cell cultures. Equal amount of protein samples were separated by SDS-PAGE, and transferred to PVDF membranes, which were blocked, and reacted with primary antibodies against Rab8a (1:1000, Abcam, Cambridge, UK), Rab11a (1 : 500, Abcam), or GAPDH (1:1000, Proteintech, Wuhan, China) according to the manufacturer's recommendations. The specific binding of primary antibody was detected by HRP-conjugated species-specific secondary antibody (Beyotime, Shanghai, China) and enhanced chemiluminescence assay.
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