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. 2016 Jun 6;6:27401. doi: 10.1038/srep27401

Figure 1. Thr4-P levels are subject to changes in mitotic cells.

Figure 1

(a) Pol II large subunit (Rpb1) carboxy-terminal domain (CTD) containing heptad repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Consensus repeats have five potential phosphorylation sites. Amino acid residues that differ from the consensus motif are depicted in red. (b) Treatment of HeLa cells with nocodazole induced a slower migrating Thr4-P-specific Pol II00 form. Cells were treated with nocodazole (20 ng/ml) for the indicated time points and extracts of adherent and shake off cells were analyzed by western blotting with mAbs specific for CTD modifications Thr4-P (6D7), Ser2-P (3E10), Ser5-P (3E8), Ser7-P (4E12), or Rpb1 (Pol 3.3). II0 and IIA represent the known hyper- and hypophosphorylated forms of the large subunit Rpb1 of Pol II; II00 represents a new slower migrating form. H3Ser10-P served as a marker for mitotic cells and actin was the loading control. WCE, whole cell extract. a, asynchronous cells. m, mitotic shake off cells. (c) Treatment of HeLa cells with 500 nM okadaic acid (OA) induced the Thr4-P-specific Pol II00 form. Cells were treated with different concentrations of OA for 90 min and whole cell extracts were analyzed by western blotting with antibodies that detected specific CTD modifications, Rpb1 or H3Ser10-P. Tubulin served as the loading control.