Ab124830

Protein was extracted using radioimmunoprecipitation assay buffer [50 mM Tris HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P40 and 0.1% sodium dodecyl sulfate] and phenylmethanesulfonyl fluoride at 4°C. Bicinchoninic acid protein assay kit (BestBio, Shanghai, China) was used to determine the concentration. Total protein (10 µg per well) was separated using 10% SDS PAGE and then transferred to a polyvinylidene difluoride membrane. The membranes were blocked using skimmed milk for 1 h at room temperature, then the blots were incubated with primary antibody against FOS like 2 AP-1 transcription factor subunit (FOSL2; 1:1,000; ab124830; Abcam, Cambridge, UK) overnight at 4°C. Following washing in 0.1% Tween PBS-T, the blots were incubated in goat anti-mouse horseradish peroxidase-conjugated secondary antibody (1:5,000; ab6789; Abcam, Cambridge, UK) for 1 h at room temperature. Proteins were detected using electrochemiluminescence (ECL kit; Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China). Image J (version k1.45; National Institutes of Health, Bethesda, MD, USA) was used to measure densitometry.

Total proteins from cells, tissues, and exosome samples were extracted using a RIPA kit (Beyotime Biotechnology, Jiangsu, China). They were separated on polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-FOSL2 (ab124830; Abcam, Cambridge, UK) antibodies at 4°C overnight, and they were then incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse immunoglobulin G at room temperature for 1 h. To identify exosome markers, primary antibodies against TSG101 were purchased from Abcam (Cambridge, UK), and primary antibodies against Hsp 70 were obtained from Cell Signaling Technology (CST, Beverly, MA, USA). The secondary antibodies were F(ab)2 fragments of donkey anti-mouse immunoglobulin or donkey anti-rabbit immunoglobulin linked to horseradish peroxidase (Jackson ImmunoResearch Laboratories, USA). Proteins were visualized using Pierce ECL Western Blotting substrate and autoradiography. Blots were analyzed using Quantity One 4.6.