Figure 5

CycT1-U7 is ubiquitinated. A. HeLa cells stably expressing myc-CycT1-U7 proteins (HeLa/myc-CycT1-U7) were treated (lanes 1 and 2) or untreated (lane 3) with Epoxomicin (50 μM) for 30 min prior to cell lysis. The Myc-CycT1-U7 proteins were immunoprecipitated with anti-Myc antibody followed by Western blot analysis with anti-Ub antibody to detect ubiquitinated Myc-CycT1-U7 proteins (upper panel). Normal mouse IgG (mIgG) was used as a negative control for immunoprecipitation (lane 1). The expression of the Myc-CycT1-U7 proteins in 10% of the input samples was also detected by Western blot analysis using anti-Myc antibody (lower panel). B. CycT1-U7 binds Tat. HeLa/Myc-CycT1-U7 cells were transfected with HA-Tat. The cells were treated (lanes 1, 3 and 4) or untreated (lanes 2, 4 and 5) with Epoxomicin (50 μM) for 30 min prior to cell lysis. The Myc-CycT1-U7 proteins were immunoprecipitated with anti-Myc antibody (lanes 4 and 6) followed by Western blot analysis with anti-HA antibody to detect Tat proteins. Normal rabbit IgG (rIgG) was used as a negative control (lanes 3 and 5). Tat proteins in the input samples (10%) were also shown (lanes 1 and 2).