Fig.3.

TEF-1 binds to the IFITM3 promoter. (A) EMSA of the wild-type probe (Fig. 1B) with SW-13 nuclear extracts. A protein/DNA complex was formed (lane 1) which was competed by a 100-fold excess of the unlabeled wild-type probe (WT). TEF-1 antibody super-shifted the complex (lane 4), while the pre-immune serum did not (lane 3). The shifted and supershifted bands are indicated by arrows. (B) EMSA was performed as above, with HeLa nuclear extracts. (C) EMSA was performed as above, with in vitro translated TEF-1. M: 100-fold excess of the mutant probe (Fig. 1B, M). (D) WT1, M1 and M2: probes used in the EMSA shown in figure 3E, lanes 1−6 (WT1: lanes 1 and 2; M1: lanes 5 and 6; M2: lanes 3 and 4). Sites A and B: primary and secondary TEF-1 binding sites, respectively, in the wild-type probe (WT). M1 and M2: site A and site B mutant probes, respectively. Mutated sites are in bold. WT2 and M3: probes used in the EMSA shown in Fig. 3E, lanes 7−10 (WT2:lanes 7 and 8; M3: lanes 9 and 10). WT2 :wild-type probe, M3: mutant probe. Mutated sites are in bold. (E) EMSA with the SW-13 nuclear extracts and in vitro translated TEF-1.The protein/DNA complex formed between the SW-13 nuclear extracts and the wild-type probe (WT1, lane 1) is supershifted by TEF-1 antibodies (lane 2). A protein/DNA complex is formed with the site B mutant probe (M2, lane 3) and is supershifted by the TEF-1 antibodies (lane 4). The complex formation is significantly reduced when the site A mutant (M1) probe was used. A very faint complex formation is observed (lane 5, arrow) that is supershifted by TEF-1 antibodies (lane 6, arrow). The protein/DNA complex formed between the in vitro translated TEF-1 and the wild-type probe (WT2, lane 7) is supershifted by TEF-1 antibodies (lane 8). The mutant probe (M3, lanes 9 and 10) did not form a specific complex. The non-specific bands in lane 9 and 10 are marked with arrows.