FoxB protein binds directly to the cis-regulatory element of the Bra gene. FoxB protein binds directly to the cis-regulatory element of the Bra gene. (A)

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FoxB protein binds directly to the cis-regulatory element of the Bra gene. FoxB protein binds directly to the cis-regulatory element of the Bra gene. (A) The 5′-upstream sequence of the Hr-Bra gene. Red boxes indicate the two putative Fox-binding sites (Fox BS1, Fox BS2) suggested in the previous study (Matsumoto et al., 2007). Thick arrow indicates the transcription initiation site (+1). (B) Schematic drawing of Hr-FoxB protein and its variants. Black box indicates the forkhead DNA-binding domain (FH DBD). (C-F) Expression of Bra at the 110-cell stage. Fertilized eggs were injected with 50 pg of control YFP mRNA (C), 7.5 or 50 pg synthetic Hr-FoxB mRNA (D), 50 pg of mRNA encoding Hr-FoxB conjugated with the VP16 transcription activation domain (E) and with the EnR transcription repression domain (F). Scale bar: 100 μm. (G) Probe sequences of Fox BS1 and its mutant (Fox BS1m). The putative consensus core sequence, which is adopted from Carlsson and Mahlapuu (Carlsson and Mahlapuu, 2002), is in red. (H) EMSA performed with the recombinant full-length Hr-FoxB protein. The shifted band decreased gradually upon addition of a 10-fold or 50-fold excess of unlabeled competitor (lanes 2, 3, respectively). The band was not observed with the mutated probe (Fox BS1m, lane 4), and was supershifted by addition of the his-antibody (lane 5). Hidehiko Hashimoto et al. Development 2011;138:2591-2600 © 2011.