PGE₂ treatment induces binding of Smad3/4 to its DNA consensus sequence and promotes phospho-Smad3 nuclear translocation. (A) Electrophoretic mobility shift assays of nuclear extracts from HUVECs treated for the indicated times with 1 μM PGE₂ (left) or 0.5 ng/mL TGFβ1 (right). Extracts were incubated with a ³²P-labeled probe corresponding to the Smad3/Smad4 consensus sequence from the PAI-1 promoter. Both stimuli transiently induced a protein-DNA complex (➤) over a similar timescale. The figure shows a representative experiment of 3 performed. (B) Electrophoretic mobility shift assay of nuclear extracts from HUVECs treated for 1 hour with 1 μM PGE₂ and incubated with the same probe as in panel A; preincubation of the extracts with antibodies (Ab) against either Smad3 (S3) or Smad4 (S4) gave rise to a supershift (➡) of the PGE₂-inducible complex (➤). The figure shows a representative experiment of 2 performed. (C) Western blot showing the nuclear content of phospho-Smad3 (pS3; top panels) in HUVEC nuclear extracts. Cells were either treated for the indicated times with 1 μM PGE₂ or 0.5 ng/mL TGFβ1 (left)_, or with a range of PGE₂ concentrations for 1 hour (right). The nuclear protein PTB-associated splicing factor (PSF) was probed as a loading control (bottom panels). Molecular weights (kDa) are shown on the right. Experiments were repeated a minimum of 3 times.