Western blotting image analysis software

The concentration of protein extracted from tissues and cells was measured using the BCA kit (Wuhan Boster Biological Technology., Ltd, Wuhan, Hubei, China). The extracted protein was then added to wells with loading buffer (30 μg/well) and boiled at 95°C for 10 min. Proteins were then separated on 10% polyacrylamide gels (Wuhan Boster Biological Technology., Ltd, Wuhan, Hubei, China) by electrophoresis for 45-70 min at a voltage of 80-120 V and with a wet transmembrane voltage of 100 mV for 45-70 min. Proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes, sealed with 5% bovine serum albumin (BSA) for 1 hour at room temperature, and incubated with Bmi-1 (1:20000 dilution; ab126783, Abcam, Cambridge, UK) and β-actin (1:10000 dilution; ab8226, Abcam, Cambridge, UK) primary antibodies overnight at 4°C. Membranes were then rinsed with Tris-buffered saline/Triton-X100 (TBST) (3 times × 5 min) and incubated with corresponding secondary antibodies at room temperature for 1 h. The membranes were washed again (3 times × 5 min) and chemiluminescence reagent was added for image development. Images were developed using a Gel Doc EZ imager (Bio-Rad, Hercules, California, USA) with β-actin as used as internal reference. The gray value of the target band was analyzed using western blotting image analysis software (Alpha Innotech, Hayward, California, USA).