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Fig. 2 Identification of Her protein binding sites within the anterior stripe element (ASE). A: Two Her protein binding sites were identified in the ASE (lanes 4-6 and 15-17). Control reactions contained unprogrammed lysate (lanes 1, 4, and 15). FlagHer12 forms a single complex with the control E-box, ASE-E1, or ASE-E2 probes (lanes 2, 5, and 16). Specificity of binding was confirmed by supershifting complexes with an anti-Flag antibody (lanes 3, 6, and 17). Mutation of the E-box in the ASE-E1 or ASE-E2 probe abolished Her12 binding (lanes 7-9 and 18-20). Sequence specific binding was confirmed by competition assays (lanes 10-14). Reciprocal competition was observed between the control and ASE-E1 oligonucleotides (lanes 11 and 13). Oligonucleotides containing mutated E-boxes were unable to compete (lanes 12 and 14). An equivalent analysis was performed for the E2 site (lanes 15-25). Unlabeled competitors were added at 100-fold molar excess. B: Her1 also binds to the E1 and E2 sites. Control reactions contained unprogrammed lysate (lanes 1, 4, 7, 10, and 13). Proteins from MycHer1 synthesis reactions formed three complexes with the control E-box, ASE-E1 or ASE-E2 probes (lanes 2, 5, and 11). However, only one of these complexes could be supershifted using an anti-Myc antibody (lanes 3, 6, and 12), indicating that the other bands are likely experimental artifacts. Mutation of the E-box in the ASE-E1 or ASE-E2 probe eliminated Her1 binding (lanes 7-9 and 13-15).