Fig 1: The effects of DMAT and TBBz on phosphorylation of RNAPII and other nuclear proteins. (A) Equal amounts of nuclear extracts prepared from untreated and inhibitor-treated cells (60 min) were separated by SDS-PAGE followed by Western blotting with anti-RNA PII CDT repeat (ab5408, Abcam) or anti-Ser2-phospho-RNAPII CDT (ab5095, Abcam). Similar patterns were obtained in 3 different experiments using different nuclear extracts. (B) Bacterially expressed recombinant hnRNP K protein (rec-hnRNPK, left panel) was phosphorylated by CK2 kinase, purified from rat liver (Sigma; C3460), with increasing concentrations (0; 0.001; 0.01; 0.1; 1.0, 10; 100 µM) of TBBz or DMAT. Assays were stopped by boiling samples in Laemmli loading buffer. K protein was separated by SDS-PAGE, and dried gels were autographed (right panel - representative gel with TBBz treatment). (C) Densitometry of the results with inhibitors are expressed as the percentage of control kinase activity and represent means ± S.D from 3 independent experiments. (D) Nuclear extracts prepared from untreated and inhibitor-treated cells were used for autophosphorylation reactions with or without 1, 10, and 25 µM TBBz or DMAT. Assays were stopped by boiling samples in Laemmli loading buffer. Proteins were separated by SDS-PAGE, and dried gels were autoradiographed (Phosphorimager) (E). Densitometrical analysis of two phosphorylated protein bands [marked on panel (D)] is shown as means ± S.D from results expressed as the percentage of controls.