Co ip assay

Co-IP assay was performed following the manufacturer's instructions (Thermo Scientific). Briefly, 10–75 μg of affinity-purified antibodies were immobilized, with anti-IgG as the control, and stored at 4°C. Then, 200–400 μl IP Lysis was added to each well of 6-well plates. Lysates were centrifuged at 13000 g for 10 min to extract proteins. For 1 mg of lysate, 40 μl of Control Agarose Resin slurry was added to a spin column to pre-clear lysate. Harvested proteins were incubated overnight with immobilized antibodies at 4°C, then separated by SDS-PAGE and transferred onto PVDF membranes for western blotting analysis. Primary antibodies included anti-P130 (1:1000, Anti-rabbit IgG, Santa Cruz) and anti-E2F4 (1:1000, Anti-mouse IgG, Santa Cruz). Secondary antibodies included Anti-rabbit IgG (1:1000, Santa Cruz) and Anti-mouse IgG (1:1000, Santa Cruz). Co-IP samples included total protein (4 μl), anti-IgG lysate combined with target protein (control group, 20 μl), anti-P130/E2F4 lysate combined with target protein (experimental group, 20 μl), anti-IgG lysate supernatant combined with target protein (20 μl) and anti-P130/E2F4 lysate supernatant combined with target protein (20 μl).

The Co‐IP assay (Thermo Scientific, Waltham, MA, USA) was used to identify interacting protein. Specific primary antibodies (10 μg) were added to 2‐mg protein and incubated overnight at 4°C. The proteins bound to primary antibodies were eluted and subjected to denaturation and WB experiments. Anti‐IgG served as a negative control.