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. 2008 Oct 20;29(1):254–265. doi: 10.1128/MCB.01030-08

FIG. 1.

FIG. 1.

PACT overexpression reverses PKR-mediated inhibition of HIV-1 LTR expression. (A) PACT reverses PKR activity on HIV-1 LTR expression in HeLa cells. HeLa cells were transfected with 0.05 μg of pGL2-LTR-Luc (lanes 1 to 7) and 1 μg of pcDNA1-PKRwt (PKR) as indicated. Cotransfection was performed with 0.05 (lane 3), 0.1 (lane 4), 0.25 (lane 5), 0.5 (lane 6), or 0.95 (lane 7) μg of pCMV-Flag-PACT. In each experiment the empty vector (pCMV-Flag) was added to reach the same amount of transfected DNA. The activation is the ratio between the Luc level in the presence of PACT and/or PKR versus LTR-Luc alone. Whole-cell extracts (150 μg) (lanes 1 to 7) were subjected to SDS-PAGE and blotted with anti-PACTeg2 and antiactin antibodies as indicated. The lower band of PACT is the endogenous form, whereas the upper band is the transfected Flag-PACT. The graph represents the average of three independent transfections ± standard error of the means. (B) PACT does not increase the RNA level of an LTR-Luc reporter gene inhibited by PKR. Cells were transfected as described in panel A. RT-PCR was performed on RNA extracted from the transfected cells as indicated in Materials and Methods. Lane M, molecular weight markers; lane C, control with no cDNA. The Luc- and the GAPDH-amplified DNAs are indicated on the right.