Total protein of exosomes, cells, and cardiac tissues were isolated using the RIPA Lysis Buffer (BosterBio, USA). Western blots were performed as previously described.27 (link) The primary antibodies used in this study were as following: Insulin-like growth factor 1 receptor (IGF-IR) (1:500), CD9 (1:500), CD63 (1: 500), Flotillin (1:1000), Tsg101 (1:1000), Rab11a (1:1000), and β-actin (1:5000) (Santa Cruz, Shanghai, China). The protein band was imaged on a Bio-Rad Chemidoc system (Bio-Rad Laboratories, USA).
Rab11a
Manufactured by Santa Cruz Biotechnology
Sourced in China
Rab11a is a small GTPase protein that functions as a regulator of intracellular membrane trafficking. It is involved in the recycling of endosomes and the transport of various cargoes between the plasma membrane, endosomes, and the trans-Golgi network.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Boster Bio (1 mentions)
Igf ir, by Santa Cruz Biotechnology (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Tsg101, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Streptavidin conjugated to fluorescein, by Vector Laboratories (1 mentions)
Vectashield hardset mounting medium with 4 6 diamidino 2 phenylindole dapi, by Vector Laboratories (1 mentions)
Biotinylated anti rabbit secondary antibody, by Vector Laboratories (1 mentions)
α tubulin, by Merck Group (1 mentions)
Rab27b, by Santa Cruz Biotechnology (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
3 protocols using rab11a
1
Exosome Protein Analysis by Western Blot
2
Immunofluorescence Staining of Signaling Proteins
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A549 and H1975 cells were cultured on a bio-coated cover slip (BD Bioscience, San Jose, CA, USA) and fixed using 4% paraformaldehyde for 20 min. Subsequently, the cover slips were incubated with the primary antibodies for 15 h at 4 °C. The primary antibodies used were as follows: EGFR (1:300, Santa Cruz Biotechnology); Src (1:300, Cell Signaling Technology); α-tubulin (1:300, Merck Millipore); Rab-11A (1:300, Santa Cruz Biotechnology). After a PBS wash, the cells were incubated with anti-rabbit secondary biotinylated antibody (1:2000, Vector Laboratories, Burlingame, CA, USA) and visualized using streptavidin conjugated to fluorescein (Vector Laboratories, Burlingame, CA, USA). For double immunostaining, the cells were incubated with anti-mouse secondary biotinylated antibody (1:2000, Vector Laboratories, Burlingame, CA, USA) and visualized using streptavidin conjugated to fluorescein (Vector Laboratories, Burlingame, CA, USA). The coverslips were mounted on microscope slides with VECTASHIELD® Hard Set™ mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Confocal fluorescence images were obtained by confocal laser scanning microscopy (LSCM, Nikon A1+, Tokyo, Japan) using an oil immersion lens.
3
Quantitative Western Blot Analysis of Rab GTPases
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Total proteins were extracted from tissues and transfected cells using lysis buffer, and the protein concentration was determined using a BCA protein assay kit. Proteins (20 µg) were subjected to 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 2% bovine serum albumin and cultured with primary antibodies against RAB27A (dilution 1:600; cat. no. sc-74586; Santa Cruz Biotechnology, Inc.), RAB27B (dilution 1:800; cat. no. DF12060; Affinity Biosciences), RAB21 (dilution 1:1,000; cat. no. sc-81917; Santa Cruz Biotechnology, Inc.), RAB11A (dilution 1:1,200; cat. no. sc-166912; Santa Cruz Biotechnology, Inc.) and RAB9A (dilution 1:1,000; cat. no. sc-71950; Santa Cruz Biotechnology, Inc.) for 60 min at 37°C. Following 3 washes with TBST, the membranes were incubated with goat anti-rabbit IgG-HRP secondary antibody (dilution 1:1,000; cat. no. sc-2004; Santa Cruz Biotechnology, Inc.) at room temperature for 30 min and then washed as aforementioned. Subsequently, specific binding was detected with the chemiluminescence (GE Healthcare Life Sciences). The detection of the chemiluminescent signal was performed in the gel documentation system ImageQuant LAS 4000 Mini (GE Healthcare Life Sciences). The intensity of the bands corresponding to the target proteins was analyzed using ImageJ 1.8.0 (National Institutes of Health).
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