p13 is stabilized by Tax. (A) 293T cells were transfected without (−) and with increasing amounts of p13 and lysates immunoblotted by anti-HA and anti-tubulin antibodies (top panel, −Tax). The +Tax (bottom panel) is the same experiment presented in Figure 1E and added here for comparison. (B) RT-PCR was performed on 293T cells transfected with vector control DNA, p13 alone, p13 and Tax, or Tax alone. Total RNA was collected 48 hours after transfection and subjected to real-time PCR using primers specific for p13 (top panel), Tax (middle panel), or actin (bottom panel). The graph represents p13 mRNA levels from 3 independent experiments. (C) 293T cells were transfected with the CMV-driven, Rex-dependent reporter plasmid pRXRE-CAT and Rex without and with increasing amounts of p13-HA. Whole-cell lysates were immunoblotted for expression of Rex, p13-HA, CAT, and tubulin. (D) To measure the half-life of the p13 protein, 293T cells were transfected with p13 in the absence or presence of Tax. The cells were treated with 10μM cycloheximide 48 hours after transfection. Whole-cell lysates were prepared at the indicated times and the protein levels were examined by Western blot analysis for p13 (anti-HA). The bottom panel is a Coomassie blue–stained gel shown as a loading control.