PGE₂ treatment does not affect STAT5 phosphorylation or DNA binding.

(A) EMSA of nuclear protein extracts of AS-E2 cells that were pretreated with or without PGE₂ (1 μM) and stimulated with or without EPO (2 U/mL) for the indicated periods. Equal amounts of nuclear protein extracts were used. (B) Supershift analysis of nuclear protein extracts of AS-E2 cells pretreated with or without PGE₂ and stimulated with (+) or without (−) EPO. For supershifts, nuclear extracts were incubated with antibodies against STAT5A (α5A) or STAT5B (α5B) or both and supershifted STAT5 is indicated by ←. As negative control, a 100-fold excess of unlabeled STAT5 (cold self) was added to binding mixture prior to gel shift analysis. (C) Western blot analysis of the same nuclear protein extracts used for panel A using antibodies against phosphotyrosine 694/699 STAT5 (αP-Y-STAT5), phosphoserine 726/731 STAT5 (αP-S-STAT5), or total STAT5 (αSTAT5). Representative autoradiograms and Western blots from 3 independent experiments are shown.