Figure 3
Association of HA-CycT1-V107E with P-TEFb interacting partners and its RNA targets. (a + b) HA-CycT1-V107E mutant does not bind TAR or 7SK snRNA in cells – HEK-293T cells were co-transfected with HA-CycT1-V107E mutant, or HA-CycT1-wild type, HIV LTR-Luciferase and pCDNA-Myc-Tat plasmids. 48 hr. post transfection, cells were lysed and immuno-precipitated (IP) with either anti-HA antibody, or control non-immune anti-human IgG. RNA was extracted from IP and input samples (1%) and was then subjected to cDNA synthesis, which was further analyzed by real time PCR using 7SK-snRNA (a) and TAR specific primers (b). Reactions were analyzed by real time PCR in triplicates and presented as fold of mean enrichment relatively to PCR results obtained for cells transfected with LTR-Luciferase alone - set to 1. Error bars show ± SEM values. (c) Association of HA-CycT1-V107E mutants with P-TEFb in cells – HEK-293T cells stably expressing either HA-CycT1-wild type or HA-V107E-CycT1 were co-transfected with Flag-Tat using lipofectamin 2000 (Invitrogen). 48 hr. post transfection, cells were lysed and subjected to IP with α-Flag antibody. IP reactions were analyzed by WB with a Cdk9 antibody. α-HA WB represents 1% of input of HA-CycT1. (d) Association of HA-CycT1-V107E mutants with P-TEFb transcription partners in cells – HEK-293T cells stably expressing either HA-CycT1-wild type or HA-V107E-CycT1 were lysed and subjected to IP with α-HA (left panel). IP reactions were analyzed by WB with the indicated antibodies.