Expression of Vpu B-HA or Vpu C-HA in MCF-7 cells results in the accumulation of p53 and P-362/366 p53. (A) Equal amounts (2 μg) of Vpu B, Vpu C, or M Vpu-HA were transfected into MCF-7 cells by Lipofectamine 2000 for 48 hours. Cell lysates were subjected to immunoblotting with indicated Abs. (B) MCF-7 cells were transfected with siRNA against GFP or β-TrcP, and immunoblot analysis was done. (C) Increasing amounts (0.5, 1, and 2 μg) of Vpu B-HA was transfected into MCF-7 cells. (D) Cycloheximide (CHX) chase to check half-life of endogenous p53, MCF-7 cells either untransfected or transfected with M-Vpu HA or Vpu B-HA, were treated with 100 μg/mL CHX and harvested at the indicated time points for immunoblotting. (E) After quantification, the signals obtained in panel D were used to calculate the p53/GAPDH ratios that were plotted with respect to treatment period. (F) Equal amounts of Vpu B, Vpu C, or M Vpu-HA were transfected into MCF-7 followed by treatment with 1 μg/mL doxorubicin for 12 hours. Immunoblotting analysis was performed with anti-p53, anti–Iκβ-α, anti–β-TrcP, anti-HA, anti–P-362/366 p53, and anti-GAPDH Abs. GAPDH was used as a loading control. This is a representative result obtained from 3 independent experiments.