Phospho p53 mapk ser15

After embedded with paraffin, tissues were deparaffinized and rehydrated with xylene and ethanol. Then, tissues were incubated in 3% H₂O₂ for 10 min to eliminate endogenous peroxidase activity. After blocked with 10% goat serum (diluted in PBS) for 10 min, tissues were incubated with primary antibody at the dilution of 1 : 50 and incubated at 4 °C overnight. Biotin‐labeled secondary antibody and horseradish phosphatase‐labeled streptavidin were then added and incubated for 30 min, followed by substrate reaction using DAB reagent. The reaction was stopped with tap water, and then, tissues were counterstained, dehydrated, and mounted. The primary antibody includes PLK4 (Proteintech, Rosemont, IL, USA, 12952‐1‐AP), phospho‐p53 MAPK (Ser15; Cell Signaling Technology, 9286T), phospho‐p38 MAPK (Thr180/Tyr182; Cell Signaling Technology, 4511), and p21(Cell Signaling Technology, 2947T).

After corresponding treatments, cells were harvested and lysed using ice‐cold RIPA buffer (C1053, Applygen Technologies Inc., Beijing, China). Equal amounts of protein samples (20 μg per lane) were separated on 10% SDS/PAGE and then transferred onto the poly(vinylidene difluoride) membrane (Millipore). The membrane was then blocked with 5% milk followed by incubating with the primary antibody overnight at 4 °C and corresponding secondary antibody for 1 h at room temperature. Immunoreactive proteins were detected using the Immobilon Forte Western HRP substrate (Millipore, Cat. No. WBLUF0100) by BLT GelView 6000 Pro (Biolight Biotechnology Co., Ltd., Guangzhou, China). The primary antibody of this experiment includes phospho‐p38 mitogen‐activated protein kinase (MAPK; Thr180/Tyr182; Cell Signaling Technology, Danvers, MA, USA, 4511), p38 MAPK (Cell Signaling Technology, 8690), p53 (Cell Signaling Technology, 2524T), phospho‐p53 MAPK (Ser15; Cell Signaling Technology, 9286T), p21 (Cell Signaling Technology, 2947T), cyclin D1 (Cell Signaling Technology, 2978T), and GAPDH (Cell Signaling Technology, 5174T).