Fig. 5.

Lysine 48 acetylation impairs CDK9 kinase activity and recovery from replication stress. (A) Western blot analysis of U2OS stable cell lines demonstrating expression of FLAG-HA CDK9 WT and K48Q. (B) FLAG-HA-CDK9 was purified from U2OS cells stably expressing an empty vector, FLAG-HA CDK9 WT, K48Q, or kinase dead D167N, treated with or without HU, and incubated in an in vitro kinase reaction with a recombinant fragment of RNA polymerase II CTD purified from bacterial cells as substrate. The reaction mixtures were separated by SDS/PAGE and immunoblotted with antiphospho-CTD Ser2, antipan-CTD, and anti-HA antibodies. (C and D) U2OS cells stably expressing an empty vector, FLAG-HA-CDK9 WT or K48Q were transfected with NT or CDK9 siRNA targeting the 3′ untranslated region, treated with 3 mM HU for 20 h (arrested), and released into nocodazole for 10 h (released). DNA content was analyzed by using flow cytometry. (D) The percentage (mean and SEM) of cells that completed DNA synthesis in three replicate experiments is shown. **P < 0.01. See also Fig. S5.