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. 1998 Aug;18(8):4537–4547. doi: 10.1128/mcb.18.8.4537

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Copyright © 1998, American Society for Microbiology

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FIG. 4 — UV irradiation induces AP1 binding activity to the uPA enhancer elements. (A) Induction of uPA 5′-TRE- and 3′-TRE-binding activities by UV irradiation: comparison with the collagenase TRE. NIH 3T3 cells were grown in 0.5% FBS for 16 h and either exposed (+) or not (−) to UVC. Nuclear extracts were prepared at 4 h postirradiation and incubated with 20,000 cpm of the indicated ³²P-labeled probes, which correspond to the 5′-TRE (PEA3/AP1_A) and 3′-TRE (AP1_B) elements of the uPA promoter and to the canonical TRE of the collagenase promoter (COL TRE). EMSAs were performed as described previously (20). The arrowhead indicates specific TRE-binding complexes. (B) Specificity of the induced uPA PEA3/AP1_A binding activity. NIH 3T3 nuclear extract induced for 4 h was incubated with the labeled PEA3/AP1_A oligonucleotide in the absence or presence of a 150-fold molar excess of unlabeled competitors (as described in Materials and Methods): PEA3/AP1_A, mut AG, mut PEA3, and ΔG (corresponding to variants of the uPA PEA3/AP1_A site), col TRE (AP1 binding site of the human collagenase promoter), AP1_B (uPA AP1_B site), cjun2 TRE (distal AP1 binding site of the human c-jun promoter) and IgκB (κB site of the Igκ enhancer). (C) Specificity of the induced uPA AP1_B binding activity. NIH 3T3 nuclear extract induced for 4 h was incubated with the labeled AP1_B oligonucleotide site in the presence of a 150-fold molar excess of the indicated competitors as described for panel B. (D) The uPA PEA3/AP1_A element binds members of the AP1 family of transcription factors upon UV stimulation. EMSAs with a labeled oligonucleotide containing the uPA PEA3/AP1_A site and 4-h-induced NIH 3T3 nuclear extracts were performed in the absence or presence of different antibodies as indicated. Extracts were preincubated with specific antibodies directed against the murine c-Jun (α cJun), the murine ATF2 protein (α ATF2), the rat myogenin protein (unspec), the murine c-Fos protein (α cFos), or no antibody (−). (E) UV-induced binding of AP1 transcription factors to the uPA AP1_B site. EMSAs with a labeled oligonucleotide containing the uPA AP1_B element were performed as described for panel D.

FIG. 4 — UV irradiation induces AP1 binding activity to the uPA enhancer elements. (A) Induction of uPA 5′-TRE- and 3′-TRE-binding activities by UV irradiation: comparison with the collagenase TRE. NIH 3T3 cells were grown in 0.5% FBS for 16 h and either exposed (+) or not (−) to UVC. Nuclear extracts were prepared at 4 h postirradiation and incubated with 20,000 cpm of the indicated ³²P-labeled probes, which correspond to the 5′-TRE (PEA3/AP1_A) and 3′-TRE (AP1_B) elements of the uPA promoter and to the canonical TRE of the collagenase promoter (COL TRE). EMSAs were performed as described previously (20). The arrowhead indicates specific TRE-binding complexes. (B) Specificity of the induced uPA PEA3/AP1_A binding activity. NIH 3T3 nuclear extract induced for 4 h was incubated with the labeled PEA3/AP1_A oligonucleotide in the absence or presence of a 150-fold molar excess of unlabeled competitors (as described in Materials and Methods): PEA3/AP1_A, mut AG, mut PEA3, and ΔG (corresponding to variants of the uPA PEA3/AP1_A site), col TRE (AP1 binding site of the human collagenase promoter), AP1_B (uPA AP1_B site), cjun2 TRE (distal AP1 binding site of the human c-jun promoter) and IgκB (κB site of the Igκ enhancer). (C) Specificity of the induced uPA AP1_B binding activity. NIH 3T3 nuclear extract induced for 4 h was incubated with the labeled AP1_B oligonucleotide site in the presence of a 150-fold molar excess of the indicated competitors as described for panel B. (D) The uPA PEA3/AP1_A element binds members of the AP1 family of transcription factors upon UV stimulation. EMSAs with a labeled oligonucleotide containing the uPA PEA3/AP1_A site and 4-h-induced NIH 3T3 nuclear extracts were performed in the absence or presence of different antibodies as indicated. Extracts were preincubated with specific antibodies directed against the murine c-Jun (α cJun), the murine ATF2 protein (α ATF2), the rat myogenin protein (unspec), the murine c-Fos protein (α cFos), or no antibody (−). (E) UV-induced binding of AP1 transcription factors to the uPA AP1_B site. EMSAs with a labeled oligonucleotide containing the uPA AP1_B element were performed as described for panel D.