Ab47763

HCC cells were lysed with a strong radioimmunoprecipitation assay buffer containing Halt^TM Protease Inhibitor Cocktail (Thermo, Waltham, MA, USA). The concentrations of the proteins in the lysate were determined with a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA). Proteins were detected on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane. The membranes were incubated overnight at 4° C with primary antibodies for retinoblastoma (phospho S780) (ab47763, Abcam, Cambridge, UK), retinoblastoma (ab181616) and GAPDH (BM1623, Boster, Wuhan, China), and then were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies for 2 hours. The proteins were detected using an Amersham Imager 600 (GE Healthcare Life Sciences, Boston, MA, USA).

Paraffin-embedded tissue sections were dewaxed and rehydrated before antigen retrieval by boiling in 0.01 M citrate buffer (pH 6.0) for 30 min. In all, 3% hydrogen peroxide was added for 15 min to remove endogenous peroxidase. Tissues were incubated with goat serum for 30 min at room temperature and then with anti-CyclinD1 (NBP2-32840, Novus, dilution 1:100), anti-ki67 (ZM0166, ZSGB-BIO, ready to use), anti-phospho-Cyclin D1 (Thr286) (STJ90457, St John’s Laboratory, dilution 1:50), and anti-phospho-Rb (Ser780) (ab47763, Abcam, dilution 1:100) antibodies at 4 °C overnight, respectively. The immunodetection was performed on the following day using DAB (GSK500710, Gene Tech) according to the manufacturer’s instructions. The staining scores were determined by two independent observers, based on both the proportion and brown intensity of the indicated protein-positive cells. The proportion of positively stained tumor cells was divided into 4 grades: (0: no positive cells; 1: <10%; 2: 10–50%; and 3: >50%). The staining intensity was recorded as follows: 0 (no staining), 1 (light brown), 2 (brown), and 3 (dark brown). The SI was calculated as follows: SI = the proportion of positive cells × staining intensity. Using this method, the expression of target protein was evaluated using the SI and scored as (0, 1, 2, 3, 4, 6, or 9), with a cut-off point of <3 versus ≥3.

Protein was extracted from the cells using RIPA lysis buffer containing protease and phosphatase inhibitors (78442, Thermo Scientific), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, transferred to polyvinylidene difluoride membranes. Membranes were incubated with primary antibodies overnight at 4 °C, followed by anti-mouse or rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (7076/7074, CST, dilution 1:1000). Afterwards, the protein–antibody complex was visualized by enhanced chemiluminescence assay (34095, Pierce). Primary antibodies against Cyclin D1 (2922, CST, dilution 1:1000), phospho-Cyclin D1 (Thr286) (3300S, CST, dilution 1:1000), GSK-3β (12456S, CST, dilution 1:1000), Rb (9309, CST, dilution 1:1000), phospho-Rb (Ser780) (ab47763, Abcam, dilution 1:1000), Lamin A/C (4777, CST, dilution 1:1000), and HRP-conjugated glyceraldehyde 3-phosphate dehydrogenase antibody (HRP-60004, Proteintech, dilution 1:5000) were used. uncropped blots are provided in supplemental information for uncropped blots and gels in supplementary information.