Hypoxia induces sequence-specific DNA binding activity by Smads.

(A) Electrophoretic gel mobility shift assays were performed using a³²P-labeled SBE probe as described in “Materials and methods.” Nuclear extracts were as follows: control normoxic HUVECs, C; HUVECs treated for 30 minutes with 12.5 ng/ml TGF-β2, T; or HUVECs exposed to hypoxia (1% O₂) for 4 hours, H. Competition assays: no competition (−); 100-fold molar excess of unlabeled SBE (100 × SBE); 125-fold molar excess of erythropoietin (125 × EPO), an HIF-1–containing oligonucleotide; 125-fold molar excess of an oligonucleotide that corresponds to the −77 to −40 bp TGF-β2 promoter (125 × TGF-β2). (B) Antibody supershifting: nuclear extracts from HUVECs were preincubated with anti-Smad2, anti-Smad3, or anti-Smad4 antibodies for 30 minutes prior to the binding reaction. Constitutive binding is indicated by the unlabeled arrows.