PGE₂-induced phospho-Smad3 nuclear translocation and DNA binding require Alk5 kinase activity. (A) Western blot showing the nuclear content of phospho-Smad3 (pS3) in nuclear extracts of HUVECs pretreated (30 minutes) with vehicle or the specific Alk5 inhibitor SB431542 (1 or 5 μM) and then treated for 1 hour with 1 μM PGE₂ or 0.5 ng/mL TGFβ1 (top panel). PSF was probed as a loading control (bottom panel). (B) Top panel: Western blot showing phospho-Smad3 content in whole-cell lysates of HUVECs pretreated as indicated with 1 μM Alk5 inhibitor and treated for 1 hour with 0.5 ng/mL TGFβ1. Bottom panel: Western blot showing the content of phospho-ERK-1,2 (pERK) in whole-cell lysates of HUVECs preincubated as indicated with 1 μM Alk5-specific inhibitor and treated for 10 minutes with 1 μM PGE₂. Molecular weights (kDa) are shown on the right. The figure shows results of representative experiments of 3 performed. A dividing line shows the grouping of parts of the same gel from which irrelevant lanes have been deleted.