FIG. 6.
The Brd4-P-TEFb interaction significantly increases in cells progressing from late mitosis to early G1. (A) Schematic representation (left) the experimental setup. HeLa cell growth was synchronized by a double-thymidine block. Ten hours after the release from the block, mitotic cells were collected by shake-off and replated in fresh medium. Cell lysates were prepared at indicated time points and subjected to anti-Cdk9 immunoprecipitation. The lysates and the immunoprecipitates were analyzed by Western blotting (right) for the various components, as indicated to the right. (B) Mitotic cells were also collected by shake-off at 0 h from the asychronized (AS) HeLa-based cell line F1C2 expressing Flag-tagged Cdk9-f and replated in fresh medium for 1 h. The levels of the indicated components in either the cell lysate or the anti-Cdk9-f immunoprecipitates were analyzed by Western blotting. (C) The enhanced Brd4-P-TEFb interaction at late M/early G1 is disrupted by the S175A mutation in Cdk9. Stable cell lines expressing Flag-tagged wild-type Cdk9 or S175A were collected by shake-off and replated in fresh medium for 1 h. The cell lysates and anti-Flag immunoprecipitates were analyzed by Western blotting as in panel B. IP, immunoprecipitation; WT, wild type; WB, Western blotting.