PGE₂-mediated Smad3 activation requires the presence of active TGFβ and MT1-MMP. (A) The graph represents the percentage of active TGFβ versus total TGFβ, measured by ELISA in the supernatants of HUVECs treated for the indicated times with 1 μM PGE_2. (B) Western blot showing the nuclear content of phospho-Smad3 (pS3) in nuclear extracts of HUVECs pretreated with a TGFβ-neutralizing antibody (αTGFβ) or a control antibody (C) before treatment for 1 hour with 1 μM PGE₂ (P). A dividing line shows the grouping of parts of the same gel from which irrelevant lanes have been deleted. The figure shows a representative experiment of 3 performed. (C) Western blot showing PGE₂- and TGFβ1-modulated phospho-Smad3 nuclear content as in panel B in HUVECs pretreated with the metalloproteinase inhibitor GM6001 or vehicle. The figure shows a representative experiment of 4 performed. (D) Western blot showing PGE₂- and TGFβ1-modulated phospho-Smad3 nuclear content as in panel B in HUVECs preincubated with an MT1-MMP–neutralizing antibody (αMT1) or a control antibody (C). The figure shows a representative experiment of 3 performed. In all cases, blots were probed with antiphospho-Smad3 antibody (pS3; top panels). Equal loading was confirmed either by a nonspecific band (panel B; nonsignificant) or by Western blot for the nuclear protein PSF (B,C, bottom panels).