After cell treatment, the medium was discarded. Supplementation of protein lysis solution (Roche) was completed for separating the total protein. Then, 50 μg of total protein was sampled on 12% polyacrylamide gel for 2-hour electrophoresis at 100 V. Afterward, the electrophoresed total protein was transferred to polyvinylidene fluoride (PVDF) membranes. One hour after being sealed with 5% skimmed milk at RT, the membranes were flushed three times (10 minutes each time) with Tris-buffered saline with Tween-20 (TBST) for overnight incubation at 4°C with the following primary antibodies (Abcam, Cambridge, UK, concentration 1:1000): Anti-Bax antibody (ab182734), Anti-Bcl-2 antibody (ab692), Anti-Cleaved Caspase-3 antibody (ab214430), Anti-p-AKT antibody (ab38449), Anti-AKT antibody (ab8805), Anti-p-PI3K antibody (ab278545), Anti-PI3K antibody (ab140307), and Anti-HIF-1 alpha Antibody (ab179483). Next, the membranes were maintained with the horseradish peroxidase (HRP)-labeled Goat Anti-Rabbit IgG (ab6205718) (concentration 1:2500) at RT for one hour after being rinsed with TBST. Following 3 washes with TBST (10 minutes/time), the membranes were exposed with enhanced chemiluminescence (ECL) chromogenic agent (Millipore, Bedford, MA, USA), and a membrane scanner was utilized for imaging [24 (link)].
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