Anti pik3cb

Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer containing 1% phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China) on ice for 30–60 min and centrifuged at 12,000 rpm for 5 min, and the pellet was discarded. The protein samples were boiled in sodium dodecyl sulfate (SDS)-loading dye for 15 min. The proteins were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto a 0.45-µm nitrocellulose membrane (Millipore, Bedford, MA, USA). The membranes were blocked with Protein Free Rapid Blocking Buffer (EpiZyme, Jiangsu, China) and subsequently probed with primary antibodies. The primary antibodies used were as follows: rabbit anti-human SET8 (#2996), anti-PIK3CB (#3011), anti-FOXO1 (#2880), anti-AKT (#4685), anti-p-AKT (#4060), and anti-BAK (#12105), anti-histone H4 (#13919), anti-BCL2 (#15071), and anti-GAPDH (#51332) purchased from Cell Signaling Technology (Beverly, MA, USA) and anti-H4K20me1 (Abcam; #ab177188; Cambridge, UK). Thereafter, the membranes were washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1.5 h at room temperature. Immunoreactive bands were visualized using an enhanced chemiluminescence detection system.

The total protein of GC cells was extracted by using RIPA lysis buffer with a protease inhibitor or phosphorylated protease inhibitor (Sigma Aldrich, Darmstadt, Germany). Western blotting was performed according to standard protocol with these antibodies and dilutions: anti-SP1 (Cell Signaling Technology, 9389S, 1:500); anti-PIK3CB (Cell Signaling Technology, 3011S, 1:800); anti-p-AKT (Cell Signaling Technology, 9271S, 1:500); anti-AKT (Cell Signaling Technology, 9272S, 1:500); anti-β-actin (Proteintech, 20536-1-AP, 1:3000). The proteins expression level was finally visualized using the Odyssey Infrared Imaging System.