Figure 3

KLF1 and KLF3 bind to CACCC boxes in the ORR1A0 LTR. Nuclear extracts were harvested from COS cells expressing KLF3 (A, B) and KLF1 (C, D) and were analyzed by EMSA using radiolabelled probes covering the four CACCC boxes in the ORR1A0 LTR promoter shown in Figure 2b. Unbound DNA probes are indicated by *. KLF3:DNA and KLF1:DNA complexes are represented by ** and ***, respectively. The identities of these complexes were confirmed by supershifting (****) with antibodies specific for KLF3 (A, B) and KLF1 (C, D). In (A and B), a radiolabelled probe encompassing a known KLF3 binding site in the Fam132a promoter [54] has been included as a positive control. In (C and D), a probe containing a CACCC-box from the β-major globin promoter, a site that is strongly bound by KLF1 [46], has been used as a positive control. In (A and B), a background band present in mock-transfected COS cells co-migrates with KLF3, but at a much lower intensity and it does not shift with the KLF3 antibody.