Investigation of Protein Interactions via Western Blot and Co-IP

Western blot was carried out as previously described^{27 (link)}. The primary antibodies used in Western blot were as follows: mouse anti-human Pontin (catalog SAB4200194; Sigma-aldrich, USA), mouse anti-human GAPDH (catalog BM3876; Boster, Wuhan, China), rabbit anti-human E2F1 (catalog 49286; Sabbiotech, USA), rabbit anti-human CDK1 (catalog 10762-1-AP; Proteintech, USA), rabbit anti-human CDK4 (catalog 12790 T; Cell Signaling Technology, USA), mouse anti-human cyclin B2 (catalog sc-28303; Santa Cruz Biotechnology, USA), mouse anti-flag tag (catalog F3165; Sigma-aldrich, USA). Co-IP was performed as previously described^{28 (link)}. In brief, cell extracts were immunoprecipitated with each 2 μg of indicated antibody (IgG, E2F1, or flag antibody) for 4–6 h and with protein A/G agarose beads (catalog No. sc-2003; Santa Cruz Biotechnology) overnight. Bound proteins were then washed, resuspended in protein sample buffer, separated by SDS-PAGE and detected by immunoblot.