Co-immunoprecipitation

For co-immunoprecipitation (Co-IP), the whole protein lysates prepared from HCT116 and HCT-8 cells were extracted in a RIPA lysis buffer. Briefly, RNF126 and p53 antibodies were first mixed with magnetic-beads (Thermo scientific, Rockford, IL, USA) for at least 4 hours. Antibody-beads complexes were then mixed with the supernatants of protein lysates with a rotator overnight. The final antibody–protein immunocomplex was stripped by boiling with 5x loading sample buffer for WB incubated with RNF126, p53, p21, and GAPDH antibodies. Input and IgG panels were used as the positive and negative control, respectively. Co-IP was repeated at least three times. For ubiquitination assays, we routinely took p53 as the Co-IP target (antibody-protein immunocomplex) that was incubated with anti-Ubiquitin antibody (Abcam) for late Ubiquitin detecting.