Biosci new flash protein any kd page

BioSci™ New Flash Protein any KD PAGE was obtained from Dakewe (Shenzhen, China). Anti-mouse IgG second antibody, goat anti-rabbit second antibody, anti-EZH2, and anti-PD-L1 antibody were acquired from Abcam (Cambridge, UK). The Annexin V -FITC/PI Apoptosis Kit was purchased from Solarbio (Beijing, China). Ten-percent fetal bovine serum (FBS) and crystal violet were obtained from Beyotime (Nantong, China).

To collect whole-cell lysates, cells were lysed in RIPA lysis buffer supplemented with a Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA), and the cell lysates were centrifuged at 12,000 rpm. The supernatant was collected, mixed with loading buffer, and boiled for 15 min to disengage the protein secondary structure. The mixture was resolved on SDS-PAGE gels (BioSci™ NewFlash Protein AnyKD PAGE; DAKEWE, Beijing, China) and then transferred onto nitrocellulose membranes (GE life, Pittsburgh, PA, USA). After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies overnight at 4 °C. The bands were probed with the appropriate secondary antibodies and detected by enhanced chemiluminescence. The following primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): PD-1 (D4W2J, 1:1000), phospho-AKT (Ser473) (D9E, 1:2000), phospho-mTOR (Ser2448) (D9C2, 1:1000), phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E, 1:2000), AKT (pan) (C67E7, 1:1000), mTOR (7C10, 1:1000), S6 Ribosomal Protein (54D2, 1:1000), β-TrCP (D13F10, 1:1000), and β-Actin (8H10D10, 1:1000), except anti-PD-L1 antibody [EPR19759] (ab213524; Abcam, UK, 1:1000).