Bicinchoninic acid assay protein assay kit

After the indicated treatment, the cells were washed twice with ice‐cold PBS (pH 7.4), and lysed with lysis buffer supplemented with protease inhibitor cocktail and phosphatase inhibitors (Roche, Basel, Switzerland) 100 μL per well of a six‐well plate. Concentration of the protein was measured by bicinchoninic acid assay protein assay kit (Bio‐Rad, Hercules, CA, USA). An equal amount of the protein (30 μg) was loaded, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and blotted onto polyvinylidene fluoride membrane (0.45 μm, GE Healthcare, Buckinghamshire, UK). The membranes were incubated with anti‐β‐actin (1:2000), anti‐AKT (1:1500), anti‐phophos‐AKT (1:800) or anti‐PI3K (1:1000), anti‐NF‐κB (1:500) and anti‐LaminB (1:1000) antibodies at 4°C overnight. After being washed three times, the membranes were incubated with the relevant horseradish peroxidase‐conjugated second antibodies (1:3000) for 3 h, and then the immune complexes were enhanced by chemiluminescence.

Protein concentration was quantified by using a bicinchoninic acid assay protein assay kit (Bio-Rad Laboratories Inc.). The protein extracts were run on 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to polyvinylidenedifluoride membranes. The membranes were blocked with Tris-buffered saline and Tween 20-containing 5% nonfat dried milk at room temperature for 2 hours, washed, and incubated with antisurvivin (Cell Signaling Technology, Inc., Boston, MA, USA) or anti-β-actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA) primary antibody at 4°C overnight. The membranes were then washed and incubated with corresponding secondary antibody conjugated with horseradish peroxidase (HRP) for 1 hour. Finally, the membranes were developed with enhanced chemiluminescence reagents (EMD Millipore, Billerica, MA, USA).