Fig. 2. Hes6 protein binds to an Enhancer of Split E (ESE) box and represses transcription at an ESE box containing promoter. EMSA of Hes6. In vitro translated protein was incubated with the oligonucleotides shown, as described in the Materials and Methods. (A) In vitro translation reactions containing either no DNA (left hand lane) or cDNA encoding Hes6 protein with an N terminal His Tag (remaining lanes) were incubated with a ³²P-labelled ESE box containing oligonucleotide. The binding reactions were carried out in the presence of unlabelled ESE box containing oligonucleotide present in 5-, 50- and 200-fold excess in the lanes shown. IgG indicates binding reaction carried out in the presence of control IgG; HIS, binding reaction in the presence of an anti-HIS tag antibody. (B) In vitro translations with no DNA or Hes6 cDNA were incubated with a ³²P-labelled ESE box oligonucleotide, as in A. Unlabelled E box and N box competitor oligonucleotides, present in 5-, 50- and 200-fold excess were added to the reaction as shown. (C) COS cells were transiently transfected with pIRES2-EGFP plasmids with inserts encoding ß-galactosidase or mouse Hes6, with a reporter vector consisting of a collagenase promoter driving expression of firefly luciferase (pCOLluc3) or pCOLluc3 with 2 ESE box motifs cloned immediately 5' of the collagenase promoter (pCOLluc3-ESE). A renilla luciferase reporter (pRL-TK) was used as a control for transfection efficiency. The values shown represent the means of four independent experiments, each performed in triplicate or quadruplicate wells, normalised to the pIRES ß-gal + pCOLluc3-ESE control. Error bars show s.e.m. *P=0.010 using a two-tailed paired t-test, comparing Hes6 with ß-gal control, when each was co-transfected with pCOLLuc3-ESE reporter.