Binding of Tβ4-induced nuclear proteins to the PAI-1/AP-1–like sequence and the AP-1 consensus sequence. EMSA was performed using labeled oligodeoxynucleotides containing the AP-1–binding sequence from the PAI-1 promoter (AP-1/PAI-1, lanes 1-6) or consensus AP-1–binding sequence (TGAGTCA) flanked by 13 bp from PAI-1 promoter (AP-1con, lanes 7-15). Nuclear extracts used in these studies were prepared from confluent EA.hy 926 cell cultures unstimulated (lanes 1 and 8) or stimulated for 2 hours either with Tβ4 (40 nM; lanes 2-5, 9-12, and 14-15) or TNF-α (10 ng/mL; lanes 6 and 13). For competition experiments, unlabeled double-stranded competitor oligodeoxynucleotides in 200-fold molar excess were added to the reaction mixtures as indicated (lanes 3-5 and lanes 10-12). Immunologic identification of Tβ4-induced DNA-binding protein was performed with mouse monoclonal antibodies specific to fos/jun family members: c-Jun phosphorylated on Ser63 (lane 14) and c-Fos (lane 15). Protein-DNA complexes were resolved on a native 5% polyacrylamide gel and detected by autoradiography. Protein-DNA complexes and supershifts with antibodies are marked by arrows. Lane 7 contains only DNA probe.