Figure 4.

Expression and binding of the TFs AP-1 and Sp1/Sp3 to the CLU basal promoter region using nuclear extracts from hCECs. (A) Schematic representation of the 50 bp segment from the human CLU promoter (-153 to -203) used as a labeled probe in EMSA that also bears putative binding sites for AP-1 (from -188 to -182 (brown)) and Sp1/Sp3 (from positions -194 to -186 and -170 to -161 (pink)). (B) Nuclear proteins (15 µg) prepared from three different populations of hCECs (Epi52, Epi70X and Epi73X) were incubated with the CLU-labeled probe and formation of DNA-protein complexes monitored by EMSA. (C) Nuclear proteins from unwounded hCECs (Epi 70X) were incubated with the CLU-labeled probe either alone (C+) or in the presence of a 150- or 500-fold molar excess of unlabeled competitor oligomers bearing the high affinity recognition target site for AP-1, Sp1/Sp3 or NFI. (D) HCECs nuclear proteins (Epi70X) were incubated with the CLU-labeled probe either alone (negative control: C+) or with antibodies that can recognize the c-Fos or c-Jun AP-1 isoforms (Ab AP-1), or Sp1 and/or Sp3 prior to separation of the DNA-protein complexes by EMSA. SSC: supershifted complexes formed upon addition of the antibodies. (E) Nuclear proteins from unwounded (Ctrl) or scratch-wounded (Wounded) hCECs (Epi 52) were incubated with the CLU probe either alone (C+) or in the presence of either a 150- or 500-fold molar excess of unlabeled competitor oligomers bearing the target site for either AP-1 or Sp1/Sp3. The positions of both the AP-1 and Sp1/Sp3 DNA-protein complexes are indicated. P: labeled probe without added proteins; U: free probe; N.S.: non-specific complex.