2.5. Western Blotting and Immunoprecipitation

HeLa cells were treated with different concentrations of sinocrassulosides VI/VII (2, 4, and 8 μM) for 12 h at 37°C. Cells were lysed using ice-cold modified RIPA buffer [50 mM Tris-HCl, 150 mM NaCl, 0.25% SDS, 1% Triton X-100, 0.25% sodium-deoxycholate, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT), and protease inhibitor cocktail (Roche)]. Proteins from the cell lysate were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, USA). The membranes were incubated with primary antibodies p16, CDK4, CDK6, cyclinD1, pRb, E2F1, and Tubulin (1 : 1000, Cell Signaling Technology, Beverly, USA) overnight at 4°C, followed by incubation with HRP-conjugated secondary antibodies (1 : 5000, Santa Cruz, CA) for 1 h at room temperature. All signals were visualized using ECL Western blotting substrate (Pierce, Thermo Fisher Scientific) according to the instructions of the manufacturer.

For immunoprecipitation, HeLa cells were treated with 4 μM sinocrassulosides VI/VII at 37°C for 12 h. Cell protein extracts (1 mg protein in 500 μl lysis buffer) were incubated with 2 μg of Rb antibodies or normal IgG at 4°C for 2 h, followed by 50 μl of protein G-agarose suspension (Santa Cruz, CA) overnight at 4°C with gentle shaking. The immunocomplexes were washed three times with fresh RIRA buffer and eluted by boiling the samples in 2x SDS-PAGE loading buffer. The proteins were electrophoresed on SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with E2F1 antibodies (0.5 μg/mL) overnight at 4°C, followed by incubation with HRP-conjugated secondary antibodies for 1 h at room temperature. All signals were visualized using ECL Western blotting substrate.