Figure 7
N-terminal domains are required for triptolide to reduce Tat steady-state levels. (A) Schematic diagram of domains of HIV-1 Tat. (B) The effects of triptolide on steady-state levels of Tat mutants. HeLa cells were transfected with plasmids encoding truncated forms of Tat in the presence of increasing concentrations of triptolide. Cells were harvested 48 h post-transfection, and protein expression levels were determined by Western blotting. (C) Schematic diagram of wild-type Tat and two mutants with domain substitution. In Tat-SFV, the basic domain was replaced with NLS from SFV capsid. In Tat-NES, the NES from HIV-1 Rev was fused to the C-terminus of Tat. (D) The effects of triptolide on steady-state levels of Tat mutants depicted in panel C. HeLa cells were transfected with plasmids encoding Tat mutants in the presence of increasing concentrations of tritpolide. Cells were harvested 48 h post-transfection, and protein expression levels were determined by Western blotting. (E) HeLa cells were transfected with pEF1-FLAG-Tat or pEF1-FLAG-TatΔ49-57 in the presence or absence of 5 nM triptolide. At 48 h post transfection, the nucleus and cytoplasm were fractionated using NE-PER nuclear and cytoplasmic extraction reagent, and protein expression levels were determined by Western blotting. C, cytoplasm; N, nucleus. (F) HeLa cells were transfected with pEF1-Tat49-57-GFP in the presence of increasing concentrations of triptolide. Cells were harvested 48 h post-transfection, and protein expression levels were determined by Western blotting.