Cleaved caspae 3

After AF cells were cultured in the conditioned medium, they were washed with sterile PBS for two times. Total protein samples were extracted by RIPA solution (Beyotime, China), and then quantified using a BCA Protein Assay Kit (Beyotime, China). After protein samples were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), they were transferred on to the polyvinylidene fluoride (PVDF) membranes (Beyotime, China). Then, the PVDF membranes were sequentially immunostained with primary antibodies (GAPDH: Abcam, ab181602; cleaved caspae-3: Cell Signaling Technology, #9664; Cleaved PARP: Cell Signaling Technology, #94885; JNK: Cell Signaling Technology, #9255; p-JNK: Cell Signaling Technology, #9252; p38 MAPK: Abcam, ab31828; p-p38 MAPK: Abcam, ab47363) overnight at 4°C and the secondary antibodies for 2 h at room temperature. After the protein bands were detected by enhanced ECL reagent (BeyoECL Plus, Betyotime, China), the immunoreactive protein bands were quantified by densitometry (Image J software, Wayne Rasband, National Institutes of Health, USA) with a loading control of GAPDH.

Antibodies against p-JNK (#9255), JNK (#9252), cleaved caspae-3 (#9661), cleaved caspae-9 (#9509), p-Ser-Pro (#2325), and p-Thr-Pro (#9391) were from Cell Signaling Technology (Beverly, MA) and used at a dilution of 1:1,000 for immunoblot analysis. Antibodies against Bax (sc-20067, diluted to 1:1,000), CD63 (sc-15363, diluted to 1:1,000), TSG 101 (sc-7964, diluted to 1:1,000), and β-actin (sc-47778, diluted to 1:2,000), were from Santa Cruz Biotechnology (Dallas, TX). Antibodies against CYP2E1 (ab28146, diluted to 1:3,000), 3-NT (sc-ab6139, diluted to 1:3,000), and iNOS (ab136918, diluted to 1:1,000), were from Abcam (Cambridge, Mass).