The K358 site is vital for K63-linked ubiquitination of p53.
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(A) A schematic of p53 site mutants. (B and C) The effect of N protein or P protein on the protein expression level of wild type and mutant p53. EPC cells were seeded in 6-well plates and transfected with 1 μg p53-Myc or p53 mutant together with 1 μg empty vector, or N-Flag (B), or P-Flag (C) for 24 h. Then the WCL were subjected to IB with the Abs indicated. (D and E) The effect of N protein or P protein on the K63-linked ubiquitination of wild type and K358R p53. EPC cells were seeded in 10 cm2 dishes and transfected with 5 μg p53-Myc or K358R-Myc, and 5 μg N-Flag (D) or P-Flag (E) or empty vector, and 1 μg Ub-K63O-HA. At 18 h post-transfection, the cells were treated with MG132 for 6 h. Cell lysates were IP with anti-Myc-affinity gels. Then the immunoprecipitates and WCL were analyzed by IB with the Abs. All experiments were repeated for at least three times with similar results.