CDK9 Ser 90 substitutions affect Tat-dependent HIV-1 transcription. (A-B) 293T cells were transfected with HIV-1 LTR-Luc expression vector along with WT CDK9, CDK9 S90A and CDK9 S90D expression vectors without (panel A) or with Tat expression vector (panel B). Cells were lysed at 24 hours posttransfection and luciferase activity was measured followed by the measurement of EGFP fluorescence, which was used for the normalization. Quantification is shown for three independent experiments. (C) Expression of CDK9 and cyclin T1. To determine the levels of CDK9 and cyclin T1 expression, 293T cells were transfected with FLAG-tagged WT CDK9, CDK9 S90A and CDK9 S90D and also co-transfected with Cyclin T1 and HIV-1 Tat expression vectors. At 48 hrs post-transfection, the cells were lysed, the lysates were resolved on 10% SDS PAGE and analyzed by immunoblotting with antibodies for cyclin T1, CDK9, and tubulin was used as loading control. Lane 1, mock-transfected control.