Thrombin induces binding of SRF and phospho-SRF to SRE-1 in primary endothelial cells.

(A) Shown is a schematic representation of the probe sequences used in electrophoretic mobility shift assays. The consensus SRE motif and potential Ets site (TTCC) in the SRE-1 probe are underlined and bolded, respectively. The mutated bases in the SRE-1 and TTCC mutant probes are represented by asterisks (*). The Ets consensus probe (Ets site in bold) is based on sequence from the human Flt-1promoter.67 (B) Electrophoretic mobility shift assays were performed with γ-[³²P]–labeled SRE-1 probe and 5 μg nuclear extract from serum-starved HUVECs treated in the absence (− Thrombin, lanes 1-12) or presence (+ Thrombin, lanes 13-24) of 1.5 units/mL thrombin. In competition assays, a 100-fold molar excess of unlabeled wild-type (lanes 2 and 14), SRE-1 mutant (lanes 3 and 15), TTCC mutant (lanes 4 and 16), or Ets consensus (lanes 12 and 24) probe was added to the reaction mixture. The closed arrow indicates specific DNA-protein complexes. The open arrows indicate nonspecific DNA-protein complexes. In supershift assays, nuclear extracts were preincubated with antibodies against SRF (lanes 5 and 17), phospho-SRF (lanes 6 and 18), Elk-1 (lanes 7 and 19), p-Elk-1 (lanes 8 and 20), SAP-1a (lanes 9 and 21), Ets-1 (lanes 10 and 22), or Ets-2 (lanes 11 and 23). The asterisk indicates the super-shifted complex.