Overexpression of HEXIM1 protein represses Tat transactivation. Panel A, Increasing amounts (10, 100 and 500 ng) of Flag-taggeted HEXIM1 were transfected into 293, cellular extracts were prepared at 48 hr after transfection and the relative levels on endogenous and exogenous HEXIM1 proteins were visualized by immunoblotting with anti-HEXIM1 antibody. Panel B, the HIV-Luc reporter (50 ng) was transfected into 293 cells in the presence of pSV-tat (50 ng) along with increasing (10, 100 and 500 ng) amounts of F:HEXIM1 and 7SK RNA (10, 100 and 500 ng), as indicated. Panel C, HEXIM1 decreases the co-operative effect of CycT1 on Tat activation in rodent cells. Chinese hamster ovary cells (CHO) were transfected with the HIV-LTR-Luc reporter (50 ng) in the presence of pSV-Tat (100 ng), lane 1, and together with CMV-hCycT1 (200 ng), lane 2, in the presence of increasing amounts of F:HEXIM1 and 7SK RNA as in panel B. Each histogram bar represents the mean of at least three independent transfections after normalization to Renilla luciferase activity to correct for transfection efficiency with the activity of the reporter without effect set to one. Standard deviations were less than 10%.