(A-C) Flat preparations; (D,E) wholemounts of two-somite stage embryos. (A,D) Wild-type embryos; (B,C,E) embryos injected with her3 mRNA encoding the full-length protein. The embryos were hybridised with islet1 (A-C) or neurog1 (D,E). Note that transcription of islet1 and neurog1 is reduced in the injected embryos. Refer to the text for further details. Anterior is towards the top. (F) Her3 protein can bind to N boxes in the neurog1 promoter. Lanes 1-3 were loaded with increasing amounts of Her3 protein (1, 3 and 5 µg, respectively) and with a labelled (5000 cpm) oligonucleotide corresponding to part of the neurog1 promoter and including two N-boxes (see NP oligonucleotide sequence in Materials and methods). A clear shift in the electrophoretic mobility of the oligonucleotide can be detected. Lanes 4-6 contain 3 µg aliquots of Her3 protein and 5000 cpm of the labelled oligonucleotide, together with increasing amounts of unlabelled oligonucleotide (5, 20 and 40 ng, respectively). Inclusion of non-radioactive homologous competitor prevents the band shift. Lanes 7-9 contain 3 µg of Her3 protein, 5000 cpm of labelled oligonucleotides, and 5, 20 and 40 ng of a heterologous competitor. Binding of the labelled probe is reduced but not completely blocked. Lanes 10-12, 13-15 and 16-18 contain 1, 3 and 5 µg of Her3 protein, and 5000 cpm of labelled oligonucleotides in which both N-boxes (lanes 10-12) [the N1 box (13-15) or the N2 box (16-18)] have been mutated (see Materials and methods). Mutation of both N-boxes reduces the affinity for Her3. As a control, lanes 19 and 20 were loaded with 5000 cpm of the wild-type (19) and mutated (20) oligonucleotides without any Her3 protein.
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