ETS activation of NAB promoters. A) Lysates of the JEG3 and S16Y (Schwann cell) lines were probed with the antibodies directed against Etv1 and Ets2, and respective blots were re-probed with β-actin antibody as a loading control. B) JEG3 cells were transfected with either the pGL3-Nab2 or pGL3-Nab1 reporter. Ets2 (25 ng), Etv1 (50 ng), or Egr2 (25 ng) expression plasmids were co-transfected as indicated in each panel. The y-axis shows the fold activation normalized to the activity of the reporter alone. Means and standard deviations of three separate transfections are shown. Average fold induction of the NAB2 promoter by Ets2 and Etv1 in 8 independent transfections was 19.5- and 7.3-fold, respectively (P =< 10-6 for both). C) Similar transfections were performed to compare ETS activation of the pGL3-Nab2 (wild type) promoter with the mutant Nab2 reporter construct in which two Egr2 binding sites are mutated (used in Figure 1c). The diagram indicates potential ETS binding sites identified by sequence analysis relative to the footprinted Egr2 binding sites in the Nab2 promoter.