Fig. 4
Identification of c-Jun in the gel-retarded complexes formed by the human liver/bone/kidney alkaline phosphatase (L/B/K ALP) 5′-12-O-tetradecanoyl-phorbol-13-acetate (TPA)-response element (TRE) and extracts from mechanically stretched human periodontal ligament (hPDL) cells. Continuous mechanical stretching was applied to hPDL cell cultures for 15 min (15′) or 30 min (30′) prior to harvesting. Whole-cell extracts were prepared and analyzed (10 µg) by an electrophoretic mobility-shift assay (EMSA), as in Figure 3. Specific activator protein-1 (AP-1)-binding activity was assayed in (A) antibody competition or (B) “supershift” experiments. Before (B) or after (A) the incubation for 30 min with anti-cJun antibody, anti-Sp1 antibody, preimmune serum or control buffer, hPDL cell extracts were incubated for 30 min with the 32P-labeled DNA probe described in Figure 3. DNA-protein complexes were resolved by electrophoresis on a 5% native polyacrylamide gel. The positions of AP-1 complexes and freely migrating probes are indicated. Note the reduction of complex formation (A) or the migration of a “supershifted” complex (B; marked by arrow) in the presence of anti-cJun antibody. “Mock” indicates a binding reaction omitting hPDL cell extracts (negative control).