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. 2012 Apr 23;7(4):e35258. doi: 10.1371/journal.pone.0035258

Figure 6. Ets-1 binds to EBE sites in the CCN2 promoter in osteoblasts.

Figure 6

Electro-mobility shift assays (EMSA) from nuclear lysates were generated from osteoblasts that were treated with TGF-β1 (5 ng/ml) for 2 hrs. (A) Nuclear protein binding to the wild type E-E-E (lanes 1–6) (this probe contains EBE # 6-8; for probe design see Figure 6) probe was assessed using 5 µg of nuclear lysates for each reaction. In some reactions, Ets-1 antibody was added at increasing concentrations (1 ug of antibody in lane 4; Two micrograms of antibody in lane 5) to test for Ets-1/probe interaction. Control antibody (2 ug) was also used (lane 6; C). In some cases, probe only (lane 1) or a molar excess of unlabeled probe (lane 3) was also used to demonstrate specificity. (B) Nuclear protein binding to the wild type S-E-T (lanes 7–14) (this probe contains EBE#5; for probe design see Figure 6) probe was assessed using 5 µg of nuclear lysates for each reaction. In some reactions, Ets-1 antibody was added at increasing concentrations (1 ug of antibody in lane 10; 2 ug of antibody in lane 11) to test for Ets-1 protein/probe interaction or Smad 3 antibody (1 ug of antibody in lane 12; 2 ug of antibody in lane 13) to test for Smad 3 protein/probe interaction. Control antibody (2 ug) was also used (lane 14). In some cases, probe only (lane 7) or a molar excess of unlabeled probe (lane 9) was also used to demonstrate specificity.