Ab202474

For Co-IP assay, magnetic beads were pre-incubated with the antibody of P73 (1:100, ab202474, Abcam). After that, the cells were lysed in with Co-IP buffer and centrifuged. The supernatant was collected and incubated with the beads overnight at 4 ℃. Finally, the beads were collected and the protein levels in the immunoprecipitates were examined by WB analysis using anti-UB antibody (1:1000, ab140601, Abcam).

Total protein was obtained by the RIPA lysis buffer (Beyotime Biotechnology Co., Ltd., Shanghai, China). According to the bicinchoninic acid analysis, the protein concentration was 1.5–2 µg/µL. After that, equal amounts of protein samples (50 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk for 15 min and covered with the diluted primary antibodies including ACTB (1:2,000, ab8226, Abcam Inc., Cambridge, MA, USA), FOSB (1:1,000, MA5-15056, Thermo Fisher Scientific), TP73 (1:1000, ab202474, Abcam), UBE3C (1:1,000, PA5-110540, Thermo Fisher Scientific), and γH2AX (1:2,000, ab81299, Abcam) overnight at 4 ℃. Later, the membranes were further incubated with secondary antibody (1:5,000, GTX213110-01, GeneTex Inc.) at room temperature for 1 h. The protein bands were developed using the enhanced chemiluminescence kit (Thermo Fisher Scientific), and the protein level relative to ACTB was analyzed by Image J.