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Fig. 5. Brn3b binds to a conserved element (SBRN3) in the Shh gene. (A) Alignment of a conserved region in the first intron of Shh in mouse, human, zebrafish and Fugu. Red letters indicate nucleotides not conserved among the species. Percentage of identity for each species when compared with the mouse sequence is indicated at the end of the sequences. The Brn3b-binding site (SBRN3) is underlined in the mouse sequence and is completely conserved in all species. The bottom shows the alignment of SBRN3 with the reported consensus Brn3b binding site. (B) Electrophoretic mobility shift assay using a GST-Brn3b POU fusion protein and a 32P-labeled oligonucleotide probe encompassing SBRN3. GST-3bPOU formed a specific complex (C), and formation of this complex was strongly inhibited by both an unlabeled Brn3b consensus oligonucleotide (100x) and the SBRN3 oligonucleotide (100x). A supershifted complex (S) was formed and retained in the loading well when anti-GST antibody was added. F is free SBRN3 probe.





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