P53 60283 2 ig

In total, 120 embryos from each sample were collected in ice-cold lysis buffer for SDS-PAGE electrophoresis, and the semi-dry transfer system was used to transfer the protein to the FVDF membrane (HATF00010, Millipore, Burlington, MA, USA), which was blocked with 5% skim milk (232100, BD, Franklin Lakes, NJ, USA) at room temperature for 1.5 h. Then, the FVDF membrane was incubated overnight at 4 °C with the specific primary antibodies (P53: 60283-2-Ig, Proteintech Group, Wuhan, China; β-actin, CW0096, CW Biotech, Beijing, China). After washing three times with TBST, 10 min each time, the FVDF membrane was incubated with the secondary antibody for 1–1.5 h. Finally, the membrane was washed with TBST three times, 10 min each time, and detected with enhanced chemiluminescence (ECL) (1705061, Bio-Rad, Hercules, CA, USA). After incubating on the NC membrane, protein bands were detected and analyzed by the protein exposure system. β-Actin was used as an internal reference.

After harvesting cells using RIPA buffer, the lysate was incubated overnight with primary antibodies MDM2 (66511-1-Ig, Proteintech), P53 (10442-1-AP, Proteintech) and Flag (#8146, CST). The protein-antibody complex was incubated with Dynal magnetic beads for 2 h and then boiled with loading buffer for 10 min. The obtained supernatant was used for western blotting analysis. Antibodies from different species were used to eliminate the effects of light and heavy chains of primary antibodies on the analysis. The following antibodies were used in western blotting: P53 (60283-2-Ig, Proteintech), MDM2 (19058-1-AP, Proteintech), and ubiquitin (sc-8017, Santa Cruz).