Fig. 7. The bHLH transcription factor MASH1 can bind and activate transcription through an E-box sequence in the I12b enhancer. (A) In a transient co-transfection assay, MASH1 activates transcription of a CAT reporter construct containing the I12b enhancer (mI12b-pBLCAT2) by a factor of about four. Values shown represent the mean relative CAT activity obtained from two independent experiments ± s.e.m. (B) EMSA using the FP5 sequence (see Fig. 2) shows binding to a combination of recombinant (r) MASH1 and E47 (lanes 2-4, arrowheads). This binding is achieved through an E-box present in the FP5 sequence. rMASH1 and rE47 were unable to bind FP5 in which the E-box was mutated (lanes 6-8; same mutation as for the transgenic mouse experiments). (C) Preincubation of rMASH1 and rE47 with an anti-MASH1 antibody completely abolishes the formation of the rMASH-rE47-FP5 complexes (lanes 3-5, arrowheads), in contrast to a binding reaction that was preincubated with the control antibody (lanes 7-9).