Ab75908

Proteins were extracted from HCC and paracarcinoma tissues by radio-immunoprecipitation assay. The protein concentration was measured using BCA protein assay reagent. Proteins were then solubilized in 4× loading buffer at a final concentration of 2 μg/μL. Next, 20 μg of protein was denatured at 95°C for 5 min followed by separation using SDS-polyacrylamide gel electrophoresis on 10% polyacrylamide gels. After electrophoresis, the proteins were transferred onto polyvinylidene fluoride membranes with a pore size of 0.45 μm. The membranes were blocked for 1 h at room temperature with TBS containing 5% BSA, followed by incubation with the primary antibody overnight at 4°C. The following primary antibodies were used in the present study: anti-MFAP4 (Abcam, ab103925, USA), anti-β-actin (Abcam, ab8226, USA), anti-p27 (Abcam, ab75908, USA), anti-CDK6 (Abcam, ab124821, USA), anti-CDK4 (Abcam, ab108357, USA), anti-pRB (CST, 8516S, USA), and anti-BCL-X (Proteintech, 10783-1-AP, China). The membranes were washed three times with TBS containing 0.1% Tween-20 (TBST) and then incubated with a secondary antibody according to the primary antibody source for 2 h at room temperature. Immunodetection was performed using an enhanced chemiluminescence (ECL) detection kit. All antibodies were diluted in TBST containing 5% BSA, and β-actin was used as internal standard.