Figure 5.

Phosphorylation of YY1 increases in nocodazole-arrested HeLa cells. (A) Endogenous YY1 was immunoprecipitated from HeLa cells incubated with radioactive orthophosphate for 2 h. Radioactive proteins were detected by Phosphorimager, and the identity of the protein was confirmed by Western blot using anti-YY1 (H-10). (B) HeLa cells transfected with pCS2(+)-Flag-YY1 or with no DNA as negative control were incubated with radioactive orthophosphate for 4 h, and WCEs were prepared. Two successive IPs were performed, a first with anti-Flag antibody (top) and the second with anti-YY1 (C-20) (bottom). Radioactive proteins were detected by Phosphorimager (left panels), and the identity of the proteins was confirmed by Western blot using anti-YY1 (H-10). (C) Flag-YY1 was immunoprecipitated from asynchronous (Asy) or nocodazole-arrested (Noc) HeLa cells, stably transfected with pCS2(+)-Flag-YY1, and separated on a 10% SDS-PAGE gel. The gel was stained with phosphospecific ProQ Diamond and subsequently with Coomassie blue. The graph represents results of quantification of five independent experiments. The synchrony of the cells was verified by FACS analysis.