Figure 2.

(A) Expression of stably integrated FLAG–CDK9 in CDK9_wt and CDK9_mt Jurkat cell clones. Parental, CDK9_wt, and CDK9_mt Jurkat cells were labeled with [³⁵S]methionine under conditions of induction (in the absence of tetracycline), and cell extracts were immunoprecipitated with anti-CDK9 or control antibodies as noted in the figure. Immunoprecipitates were analyzed by SDS/PAGE and visualized by autoradiography. As indicated, the stable FLAG–CDK9 transgene product migrates slightly higher than the endogenous CDK9. Cyclin T1 and polypeptides (of 105, 55, and 50 kDa) previously reported to coimmunoprecipitate specifically with anti-CDK9 antibodies (Santa Cruz Biotechnology) are indicated. Polypeptides immunoprecipitated by control IgG are also noted (ns). (B) Constitutive expression of FLAG–CDK9 in CDK9_wt and CDK9_mt cells. Cell extracts from [³⁵S]methionine-labeled CDK9_wt and CDK9_mt cells were immunoprecipitated with anti-CDK9 antibodies and analyzed as described above. Extracts were prepared from CDK9_wt and CDK9_mt cells both in the presence (+) and absence (−) of induction as indicated. (C) Extracts from CDK9_mt cells have reduced levels of CDK9-dependent CTD kinase activity. Extracts from parental, CDK9_wt, and CDK9_mt cells were immunoprecipitated with anti-CDK9 antibodies. Immunoprecipitates were washed and assayed for pol II CTD kinase activity as described (5) by using a CTD peptide with four heptapeptide repeats as substrate. Immunoprecipitations were normalized by both cell number and protein concentration, and the level of CTD kinase activity in the CDK9_wt and CDK9_mt cells is expressed as a percentage of the activity recovered from parental Jurkat cells. Nearly identical results were obtained in three independent experiments. (D) Tat-dependent transcription is impaired in CDK9_mt cells. An HIV LTR–luciferase reporter was transfected either alone or together with a Tat expression vector into CDK9_wt and CDK9_mt cells. An average of four independent experiments performed in triplicate is presented. (Note: The experiments shown in A were performed with three times as many cell equivalents as those shown in B.)