FIG. 4.

Activation of P-TEFb by Tat. In vitro kinase reactions were performed by using P-TEFb purified by immunoprecipitation from HeLa nuclear extract or CDK9 in transcription elongation complexes arrested by LacR. The kinase reactions were performed using a peptide carrying 5 heptad repeat sequences (CTD5) as the substrate. Reactions were performed in the presence of 1 μCi of [γ-³²P]ATP and in the absence (−) or presence (+) of 2 μM cold ATP, 2.5 mM MnCl₂, and 20 ng of Tat. The phosphorylated peptides were separated by SDS-4 to 20% PAGE, transferred onto nitrocellulose membranes, and detected by autoradiography.