Anti cdk2 antibody

RIPA Cell Lysate (Beyotime, Shanghai, China) was used to lyse SMSCs. Then, we centrifuged the lysate cells at 12,000 rpm for 10 min and collected the supernatant. BCA protein quantification kit (Beyotime, Shanghai, China) was used to determine protein concentration. Next, we subjected the protein sample to polyacrylamide gel electrophoresis, and then transferred it to PVDF (Solarbio, Beijing, China). We used 5% skimmed milk powder to seal the PVDF membrane, which was closed at 25 °C for 1.5–2 h. We then added the primary antibody and incubated it overnight at 4 °C. The next day, we used 1X TBST (Solarbio, Beijing, China) to wash the PVDF membrane. After wash-over, we incubated cells with the secondary antibody at 25 °C for 1 h. We used Electrochemiluminescence (ECL) for imprinting display. The ChemDoc^TMTouch Imaging System (Bio-Rad, Hercules, CA, USA) was used for relative expression of the protein obtained. The antibodies used in this study and their dilution ratios are as follows: Anti-CDK2 antibody (Abcam, Cambridge, UK, 1:1500), Anti-MYHC antibody (Affinity Biosciences, Cincinnati, OH, USA, 1:1000), Anti-GAPDH antibody (Abcam, Cambridge, UK, 1:1000), Goat Anti-Rabbit IgG H&L(HRP) (Abcam, Cambridge, UK, 1:5000), Rabbit Anti-Mouse IgG H&L(HRP) (Abcam, Cambridge, UK, 1:5000).

The human LC cell HepG2, Hep3B, and Huh-7 cell lines were purchased from China Infrastructure of Cell Line Resources (School of Basic Medicine Peking Union Medical College, China) and cultured in Dulbecco’s modified Eagle medium containing 10% (v/v) FBS and 100 U/ml streptomycin/penicillin at 37°C and 5% CO₂. Cell Counting Kit-8 (CCK-8) reagent was purchased from Dojindo (Japan). Hoechst 33,342 reagent was purchased from Beyotime (China). An Annexin-V FITC apoptosis detection kit (#556547) and BrdU cycle detection kit Part A (#559619) were purchased from BD Biosciences Pharmingen (United States). Antibodies against Bcl-2, Bax, Caspase 9/p35/p10, cyclin D1, and beta-actin were purchased from Proteintech (United States). Antibodies against cyclin A2, cyclin B1, p-Akt(S473), p-Akt(T308), pan-AKT, p-ERK (T202/Y204), and pan-ERK were purchased from Cell Signaling Technology (United States). Anti-CDK2 antibody was purchased from Abcam (United Kingdom). HSG was provided by Beijing Kangrentang Pharmaceutical Co., Ltd. The concentration of the prepared HSG mother solution was 0.1 g/mL. For the biological experiments, the 0.1 g/mL stock decoction could be subjected to 0.22-µm filter-sterilization and dilution as needed.