Ab126783

Total proteins were extracted from using RIPA buffer (Beyotime, Shanghai, China). BCA protein assay kit (CoWin Biotechnology) was used to examine protein concentrations. Protein was separated by SDS-PAGE, and transferred onto PVDF membrane. Membranes were incubated with following primary antibodies RNF220 (ab69357, 1:700; Abcam, Cambridge, MA, USA), SOX2 (ab92494, 1:1500; Abcam, Cambridge, MA, USA), OCT4 (ab17929, 1:1000; Abcam, Cambridge, MA, USA), NANOG (ab109250, 1:5000; Abcam, Cambridge, MA, USA), USP22 (ab195289, 1:2000; Abcam, Cambridge, MA, USA), BMI1 (ab126783, 1:30,000; Abcam, Cambridge, MA, USA), p65 (ab32536, 1:10,000; Abcam, Cambridge, MA, USA), PCNA (ab18197, 1:5000; Abcam, Cambridge, MA, USA), and β-actin (ab8227, 1:3000; Abcam, Cambridge, MA, USA) overnight at 4°C. Then, membranes were incubated with HRP-conjugated goat anti-rabbit immunoglobulin G secondary antibody (ab205718, 1:3000; Abcam). β-actin was used as an internal control to normalize the analyzed samples [16 (link)].

Human liver cancer cell lines, HepG2, PLC, and HepB3 were obtained from American Type Culture Collection (Manassas, VA, United States). Huh7 was obtained from the Japan Society for the Promotion of Science (Tokyo, Japan). These cells were maintained in 1,640 medium (Gibco, Thermo Fisher, Waltham, MA, United States) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone) and penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO2.
The reagents used in this study were: Antibodies of anti-GINS1 (ab181112), anti-BMI1 (ab126783), and anti-RAS (ab52939) were purchased from Abcam (Cambridge, United Kingdom). KLF4 (D1F2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; D16H11) were obtained from Cell Signaling Technology (Danvers, MA, United States).

Paraffin sections (8 μm) were stained with antibodies against GIPC2 (sc-515441, Santa Cruz) or BMI-1 (ab126783, Abcam) at a 1:100 dilution. The sections were incubated with biotinylated IgG (1:200, ZSGB-Bio Co.) at 37 °C for 20 min, followed by incubation with HRP-labeled streptavidin at 37 °C for 15 min. Color development proceeded for 5 min at room temperature using diaminobenzidine, followed by rinsing with distilled water. Counterstaining was performed with hematoxylin. The immunohistochemistry (IHC)-staining intensity was classified based on the quantized values from Image J, as follows: +1, <1.00 positive staining; +2, 1.01–5.00 positive staining; +3, >5.01 positive staining.

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).

The indicated cells were lysed, and then immunoprecipitated by anti-BMI1 (Abcam, Cat. No. ab126783) and anti-p53 (Abcam, Cat. No. ab1101) antibodies. Then the immune complexes were detected by anti-BMI1 (Abcam, Cat. No. ab126783) and p53 (Abcam, Cat. No. ab1101) antibodies, and visualized with an ECL analysis system.