Fig. 3

Mechanical stretching of human periodontal ligament (hPDL) cells stimulates the binding of a complex to the human liver/bone/kidney alkaline phosphatase (L/B/K ALP) 5′-12-O-tetradecanoyl-phorbol-13-acetate (TPA)-response element (TRE). Continuous mechanical stretching was applied to hPDL cell cultures for the indicated times in min (0′, 15′, 30′) prior to harvesting. Whole-cell extracts were prepared and equal amounts (5 µg) analyzed for activator protein-1 (AP-1)-binding activity by an electrophoretic mobility-shift assay (EMSA). After incubation at room temperature for 30 min with the ³²P-labeled DNA probe, equilibrated DNA-binding reactions were electrophoresed on a 5% native polyacrylamide gel. The gel was dried and then subjected to autoradiography. The positions of specific DNA-protein complexes and freely migrating probes are indicated. The sequence of the oligonucleotide used as probe is shown. The atypical TRE is highlighted in bold. “Mock” indicates a reaction mixture incubated in the absence of hPDL cell extracts (negative control).