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. 2012 Oct 24;7(10):e47862. doi: 10.1371/journal.pone.0047862

Figure 4. Sp1 and possibly GATA1 bind concomitantly to the Ccna1 promoter and suppress its activity in vivo.

Figure 4

(A) EMSA assay in nuclear extracts from indicated cell lines using radioactively labeled probe −290/−120 bp of the Ccna 1 promoter shows specific DNA-protein complex formation (arrows). Complex formation was significantly reduced by the addition of 100-fold molar excess of unlabeled DNA competitor containing an intact Sp1 site while cold competitor containing mutant Sp1 recognition site failed to disrupt the binding. (B) Firefly luciferase reporter assay reveals binding to both Sp1 and GATA1 sites suppresses activation of the Ccna1 promoter (constructs shown schematically to the left). Sp1 and GATA1 binding sites were marked as an open ellipse and black diamond, respectively. Crosses (“X”) indicate mutated binding sites. As an internal control, pRL-CMV expressing Renilla luciferase under the CMV promoter was co-transfected in each assay. Bars show the average luciferase activity of three independent experiments, normalized with respect to the Renilla luciferase activity.