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. 2015 Jan 13;6(3):1446–1461. doi: 10.18632/oncotarget.2735

Figure 5. TOPK can phosphorylate C2H2 linker sequences in vitro.

Figure 5

(A) Purified/recombinant active GST-TOPK was incubated in kinase buffer with purified GST-tagged: YY1(ZNF), Aiolos (L1), TIP20(L2), or Bcl6(L5). The kinase reactions were analyzed by Western blotting with anti-HpTGEKP and anti-GST antibodies. (B) Whole cell extracts prepared from HeLa cells arrested in S-phase (by thymidine) or mitosis (by nocodazole) were incubated in kinase buffer with or without purified/recombinant active GST-TOPK. The kinase reactions were analyzed by Western blotting with anti-HpTGEKP, anti-TOPK, and anti-Tubulin antibodies. (C) EMSA testing of the kinase reactions analyzed in (B) with radioactively labeled double-stranded DNA oligonucleotides comprising the consensus binding sites for YY1 and Sp1. The specificity of the YY1 and Sp1 shifts were confirmed by supershifting with their respective antibodies. The kinase reactions were also Western blotted and probed with YY1 and Sp1 antibodies to confirm the equal levels of the proteins in all reactions and that no degradation occurred during the kinase reaction.