Figure 3

Levels of 7SK snRNA and HEXIM1 and association with P-TEFb. (A) Total RNA was isolated from DMSO control and prostratin-treated resting CD4⁺ T cells. Northern blots were performed to measure 7SK snRNA and U1 snRNA levels; amounts of 7SK and U1 snRNA were quantified using a Phosphoimager and are shown below each panel. Levels of 7SK snRNA were normalized to U1 snRNA. (B) Immunoprecipitations were performed using α-CDK9 antibodies with extracts from control and prostratin-treated cells. CDK9 levels present in a portion of immunoprecipitates were examined by immunoblots (IP-Immunoblot). RNA was extracted from the remaining protein of immunoprecipitates and the levels of 7SK snRNA were determined by Northern blots (IP-Northern blot). Amounts of 7SK snRNA were quantified by a PhosphoImager and normalized the amounts of CDK9 present in immunoprecipitates. (C) Cell extracts were prepared from resting CD4⁺ T cells cultured in DMSO or prostratin for 48 hours to examine the levels of HEXIM1 and β-actin. Protein levels were quantified by the Densitometer and are shown below each panel. Levels of HEXIM1 were normalized to β-actin levels. (D) Immunoprecipitations were performed using antiserum against CDK9 with extracts adjusted to precipitate equivalent amount of CDK9. Immunoprecipitation products were subjected to immunoblot analysis to evaluate the levels of HEXIM1 associated with CDK9. Levels of HEXIM1 were normalized to CDK9 levels in immunoprecipitates.