Effect of Vpu expression on β-TrcP interaction with and ubiquitination of p53. (A) HEK 293T cells were cotransfected with plasmids encoding GST-p53 and either Vpu B, Vpu C, or M Vpu-HA for 48 hours as described in “Cell culture, transfections, and immunoblot analysis.” Cell lysates were used to pull down GST-p53–associated proteins with the use of Glutathione beads, and the resulting eluent was used to measure GST-p53–bound β-TrcP by immunoblotting with anti–beta]-TrcP Ab. Total cellular β-TrcP levels were not altered on Vpu expression. (B) HEK 293T cells were cotransfected with His-ubiquitin (His-Ub) and either Vpu B, Vpu C, or M Vpu-HA. After 36 hours cells were treated with MG132 for 8 hours followed by lysis in denaturation buffer, and then total ubiquitinated proteins were pulled down with the use of Ni-NTA beads, and p53 ubiquitination was checked by immunoblotting with anti-p53 Ab. (C) HEK 293T cells were cotransfected with His-Ub, and an increasing dose (0.5, 1, and 2 μg) of either M-Vpu or Wt Vpu B-HA and p53 ubiquitination was checked as described in panel B.