Faslg

The treated cells in each group were washed twice with ice-cold PBS and then lysed in 0.5 ml of lysis buffer for 20 min at 4°C. Next, the lysates were centrifuged for 10 min at 12,000 g and 4°C, and the supernatants were collected. The protein concentration in each supernatant was determined using a BCA Protein assay kit (Thermo Fisher Scientific, Inc.). Next, an equal amount of protein from each supernatant was separated by 10% SDS-PAGE, and the protein bands were transferred onto polyvinylidene fluoride (PVDF) membranes, which were subsequently blocked with skim milk. The membranes were then incubated with primary antibodies against cleaved-caspase-3 (CST, Danvers, MA, U.S.A., 9654s), IL-2 (CST, D7A5), IL-6 (CST, D3K2N), P-p65 (CST, 93H1), p-65 (CST, D14E12), TNF-α (CST, 3707), FASLG (Abcam, Cambridge, MA, U.S.A., ab15285), and GAPDH (CST, 14C10) overnight at 4°C; After which, the membranes were incubated with horseradish peroxidase-conjugated anti-IgG (Santa Cruz Biotechnology, Dallas, TX, U.S.A.) as the secondary antibody. The immunostained protein bands were visualized using an enhanced chemiluminescence (ECL) detection kit (Pierce, Rockford, IL, U.S.A.).