Protease and phosphatase inhibitor cocktail for general use

For the co-immunoprecipitation (Co-IP) experiment, HEK293T cells were co-transfected to express Myc-tagged CDK1 or (and) Flag-tagged ΔNp63α. Cells were lysed using cell lysis buffer for Western and IP (Beyotime) with protease and phosphatase inhibitor cocktail for general use (Beyotime) on ice for 10 min. Lysates were cleared by centrifugation at 13,000 rpm for 15 min. Protein concentration was determined using the Bradford protein assay reagent (Coolaber). Then, 2 mg of total proteins from cell lysates were incubated with anti-Flag or normal mouse IgG as control at 4 °C for 8 h, and the immune complexes were precipitated with protein A + G-agarose at 4 °C for 2 h. The immunoprecipitates were washed with lysis buffer, separated by SDS-PAGE and subjected to IB as described previously [79 (link),80 (link),82 (link)].

Radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China; P0013B) and protease and phosphatase inhibitor cocktail for general use (Beyotime, China; P1045) were used to obtain protein samples. Protein samples were denatured at 100°C for 10 min. Electrophoresis was started at 80 V to concentrate the protein and 120 V to separate the protein, then the transfer was conducted for 1 h under 300 mA. After blocking for 1 h with 5% non-fat milk (BD Biosciences, United States; 232100), the membranes were incubated overnight with primary antibodies BANP (ABclonal, China; A7595) and DHTKD1 (Santa Cruz Biotechnology, Inc., United States; sc-398620) at 4°C. After washing away the primary antibody with Tris-buffered saline Tween-20 (TBST), the secondary antibody (Abmart, China; M21003) was incubated for 1 h at room temperature. The bands were detected in automatic chemiluminescence and fluorescence analysis system (Tanon, China) by adding enhanced chemiluminescent reagent (NCM Biotech, China; P2200).