N protein of SVCV decreases K48-linked ubiquitination of p53.

(A) Effects of inhibitors on N protein-mediated destabilization of p53. EPC cells were seeded in 6-well plates overnight and transfected with 1 μg p53-Myc and 1 μg empty vector, or N-Flag. At 18 h post-transfection, the cells were treated with the indicated inhibitors for 6 h prior to being harvested for IB analysis of WCL with the anti-Myc, anti-Flag, and anti-β-actin Abs. (B) The N protein-induced p53 declination was rescued by MG132 in a dose-dependent manner. EPC cells were seeded in 6-well plates overnight and transfected with 1 μg p53-Myc and 1 μg N-Flag or empty vector. At 18 h post-transfection, the cells were treated with DMSO or MG132 (10, 20, or 40 μM) for 6 h. Then the WCL were subjected to IB with the Abs indicated. (C) SVCV infection suppresses the ubiquitination of p53. EPC cells were seeded in 10 cm2 plates and transfected with 5 μg p53-Myc and 1 μg HA-Ub. At 24 h post-transfection, the cells were uninfected or infected with SVCV (MOI = 10) for 18 h, then 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 indicated. (D) N protein decreases the ubiquitination of p53. EPC cells were seeded in 10 cm2 dishes and transfected with 5 μg p53-Myc, 5 μg N-Flag or empty vector, and 1 μg HA-Ub. At 18 h post-transfection, the cells were treated with DMSO or 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 indicated. (E) N protein negatively regulates the ubiquitination of p53 in a dose-dependent manner. EPC cells were seeded in 10 cm2 dishes and transfected with 5 μg p53-Myc, 5 μg empty vector, or N-Flag (5, 2.5, or 1 μg), and 1 μg HA-Ub. 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 indicated. (F) N protein decreases K63-linked ubiquitination of p53. EPC cells were seeded in 10 cm2 dishes and transfected with 5 μg p53-Myc, 5 μg N-Flag or empty vector, and 1 μg Ub-K48O-HA or 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.