Figure 5.

Endogenous Brm is required for Tat-mediated transactivation of an integrated HIV-1 LTR. (A) HeLa LTR-Luc cells were transfected twice with siRNA specific for Brm or PCAF or siRNA control. Levels of Brm, PCAF and tubulin were determined by Western blot analysis. Tat-mediated transactivation was analyzed 24 h after post-transfection with increasing amount of Tat-Flag expression vector (10, 30 or 100 ng of DNA plasmid). Fold Tat transactivation was calculated relative to transfection in the absence of Tat expression vector and normalized to Renilla activity from the TK promoter as internal control. The mean relative luciferase activities (plus standard errors) obtained from three independent transfection experiments are shown. (B) HeLa LTR-Luc cells were cotransfected twice with an siRNA specific for Brm (Brm2) or siRNA control and an HA-Brm-expressing vector. Levels of endogenous Brm, transfected HA-Brm and tubulin proteins were analyzed by Western blot. The Tat-Flag expression vector was cotransfected during the second round of transfection. The luciferase activity was determined 48 h later, and normalized to Renilla. The mean relative luciferase activities (plus standard errors) obtained from three independent transfection experiments are shown. (C) HeLa LTR-Luc cells were transfected twice with siRNA control or Brm-specific siRNA. After the second round of transfection, cells were treated either with TSA (100 ng/ml), PMA (10⁻⁸ M) or TNF-α (10 ng/ml). After 16 h, luciferase activity was measured in total lysates. The results of three independent experiments are shown. Levels of Brm and tubulin proteins were determined by Western blot analysis using anti-Brm or anti-tubulin antibodies (bottom panel, lanes 1 and 2).