Ab97694

Proteins from serums or cells were isolated by RIPA buffer (Millipore) and quantified by a BCA protein assay kit (Tiangen, Beijing, China). Next, equal amounts of protein were separated via sodium dodecyl sulfonate-polyacrylamide gel (Solarbio) electrophoresis and then transferred onto polyvinylidene difluoride membranes (PVDF, Beyotime). After blocked with 5% defatted milk for 1 h at indoor temperature, the membranes were incubated with primary antibodies against OXSR1 (1:2000; ab97694; Abcam), CD9 (1:1000; ab263019; Abcam), CD63 (1:1000; ab134045; Abcam) and GAPDH (1:10,000; ab181602, Abcam) overnight at 4 °C. Then, the proteins on the membrane were probed with the secondary antibodies (1:20000; ab205718 or ab205719; Abcam) at 37 °C for 2 h. The protein blots were visualized using BeyoECL Star Kit (Beyotime).

Protein lysates were loaded onto 4–12% BIS-Tris Protein gels (NP0336BOX; Thermo Fisher Scientific) for electrophoresis followed by transfer onto a PVDF membrane (MILIPVH00010; Merck Millipore). Membrane was blocked with 5% (vol/vol) skim milk for 1 h at room temperature, incubated overnight with a 1:1,000 dilution of rabbit anti-OXSR1 (ab97694; Abcam) followed by incubation with a 1:5,000 dilution of a donkey anti-rabbit IgG HRP antibody (AP182P; Merck Millipore) and 1:5,000 dilution of mouse anti-GAPDH (ab8245; Abcam) antibody, followed by incubation with a 1:5,000 dilution of a donkey anti-mouse IgG HRP antibody (AP192P; Merck Millipore). Protein detection was performed using SuperSignal West Pico PLUS (34579; Thermo Fisher Scientific) and imaged on a Bio-Rad ChemiDoc Imaging System.