Brg1 is recruited to the snail2 promoter in developing zebrafish. A: Wnt signaling pathway is intact in Brg1-MO-injected embryos. The cDNAs were prepared from embryos at indicated times and the expression of Wnt1, Wnt4, Wnt8b, Wnt10b, Frizzled2, β-catenin, LEF1, TCF1, Engrailed2, Snail2, Brg1, and β-Actin was determined using specific primers. Note that the expression analyses of the indicated cDNAs in the wild-type are similar to previous reports (Thisse et al.,[1995]; Dorsky et al.,[1999]; Bellipanni et al.,[2006]) and zfin.org. B: Immunoblot analyses of human Brg1 in developing zebrafish. Plasmids containing human Brg1 cDNA (pcDNA3-Brg1) (De La Serna et al.,[2000]) were injected into one-cell stage embryos. At 22 hpf, lysates were prepared and used to detect Brg1 expression using antibody to human Brg1. Note that we injected plasmids containing human Brg1 because the commercially available Brg1 antibody does not recognize the zebrafish Brg1, although there are high levels of sequence homology between Brg1 cDNA from zebrafish and other organisms. Hela cell lysate was used as a positive control. Lane termed "negative control" represents lysates prepared from zebrafish injected with empty plasmids. β-actin was used as a loading control. C: Human Brg1 efficiently rescues Brg1-MO-injected zebrafish embryos. One-cell stage embryos were left uninjected (top), or injected with Brg1-MO (middle) or Brg1-MO and plasmids containing human Brg1 (50 ng/μl) (bottom). Embryos were photographed at 25 hpf. D: Snail2 promoter regions a and b contain 3 TCF/LEF binding sites (at -1,796 bp and -2,070 bp in a, and at -2,682 bp in b) that were PCR amplified (primers location are indicated by arrows) in ChIP assays. E: Embryos were injected with control plasmids or plasmids containing pcDNA3-human Brg1 at one-cell stage. At 12 and 22 hpf, embryos were collected and prepared for ChIP assays as described in Experimental Procedures. Immunoprecipitated chromatin (or total genomic DNA in the case of input) from each group was PCR amplified for the indicated promoter fragments. Top panels represent where antibody to Brg1 was used to immunoprecipitate chromatin from embryos injected with plasmids containing human Brg1. Middle panels represent where antibody to Brg1 was used to immunoprecipitate chromatin from embryos injected with control empty plasmids. Bottom panels represent PCR products from total genomic DNA input.
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