Stable expression of CDK2-directed shRNA induces 7SK RNA expression. (A-C) Effect of CDK2 KD on small and large P-TEFb complexes. Lysates from 293T and 293T-CDK2 KD cells sequentially extracted with low and high salt buffers, were analyzed by immunoblotting for CDK9, cyclin T1, Hexim 1, eIF-2α and RNAPII. Panels B and C show average results from three experiments. (D) CDK9 inhibitor ARC prevents CDK9 with association with large P-TEFb complex. Lysates from 293T cells untreated or treated with 10 μM ARC and extracted with low salt buffer (10 mM) or high salt buffer (450 mM) were resolved on 10% SDS-PAGE and analyzed by immunoblotting for CDK9 and tubulin. Average CDK9 expression adjusted to tubulin from two separate experiments is shown. (E) CDK2 KD increases the amount of 7SK RNA associated with CDK9. RNA isolated from co-immunoprecipitates with anti-CDK9 antibodies or control IgGs from 239T or 293T-CDK2 KD whole cell lysates was reverse transcribed and analyzed by semi-quantitative (30 cycles, upper panel) or real-time PCR (lower panel). Results are presented as numbers of copies of 7SK RNA. (F) CDK2 KD increases total 7SK RNA amount. 7SK RNA was analyzed in whole cell lysates of 293T or 293T-CDK2 KD cells by real-time PCR using 7SK expression vector as control. Results are presented as numbers of copies of 7SK RNA. (G) CDK2 KD increases 7SK RNA in the large complex fraction. 7SK RNA was analyzed by real-time PCR using U6 RNA as reference. Quantification is shown in triplicates.