Es at ,2 mg per 16107cells. For ChIP-Seq, sheared chromatin was treated essentially as described [27] and converted to sequencing library for massively parallel sequencing on the Illumina GA-II platform. Sequencing was carried out in the UCSD BIOGEM Core facility. Analysis of resulting sequence reads performed in the Homer package [27] identified 13,765 Znf423 peaks at a calculated false discovery rate ,0.001. Tags were normalized to number of mapped reads for visualization in the UCSC Genome Browser as a custom track.Quantitative PCRPCR primers (Supplemental Table S1) were designed using Primer3 online tool [28]. Real-time PCR amplification was quantified by stimulated fluorescence of SYBR green dye on a Bio-Rad CFX-96 instrument. Relative quantification of ZNF423 among neuroblastoma lines compared expression in each sample to GAPDH as a conventional control by the DDCt method. For quantitative RT-PCR from mouse tissue and P19 cells, values were normalized to the geometric means of Gapdh, Pitpna, and Ppig reference genes and expressed as 22Ct(gene)/Ct(reference). Quantitative PCR from ChIP samples used either a pre-immune IgG mock ChIP or input fraction as indicated for relative quantification among samples.Materials and Methods AntibodiesZfp423 antibodies E20 and D16 were obtained from Santa Cruz Biotechnology (Figure 3A 16985061 ). Additional custom antisera were raised in rabbit against His-fusion protein expressing either residues 1?80 or 247?07 relative to human ZNF423 reference sequence NP_055884.2 and affinity-purified against the immunogen. ChIP experiments reported here used serum against 247?07 (Figure 3G ); serum against residues 1?80 performed less robustly in ChIP assays and was not considered further. EBF antibodies were a gift from Dr. Randall Reed (Figure 3A,B) or purchased from Santa Cruz Biotechnology (H300, Figure 3C ). SMAD antibodies A4 and H552 were obtained from Santa Cruz Biotechnology. Western blots were developed with infraredconjugated secondary antibodies (Rockland), detected on a LiCor Odyssey Imaging Station, and quantified in the ImageJWestern blotsHomogenized tissue, whole cell, or nuclear protein extracts were Title Loaded From File prepared in ice-cold RIPA buffer with protease inhibitor cocktail (Sigma), 10 mM DTT, 10 mM sodium orthovanadate, 8 M urea and treated with 100 U Benzonase nuclease (EMD) until minimize viscosity. Extracts were incubated in a sample buffer (50 mM Tris pH 6.8, 2 SDS, 0.1 Bromophenol blue 10 Glycerol, 33 mM DTT, 0.1 M b-mercaptoethanol, 8 M urea atZfp423 Binds Autoregulatory SitesFigure 5. Zfp423 overexpression represses intron 5 Title Loaded From File enhancer activity in P19 cells. (A) pGL4 reporter with the intron 5 enhancer was similarly active when co-transfected with shRNA directed against Zfp423 or a control. A similar plasmid with a region encompassing the intron 3 binding site had no activity above the pTAL minimal promoter. (B) Co-transfection with a plasmid expressing FLAG-tagged human ZNF423 reduced expression of the intron 5 reporter relative to a pcDNA vector control. This effect did not occur between paired samples with the Zfp423 consensus motifs mutated (intron5m). (C) An independent series of co-transfection assays indicates Ebf1-dependence of the intron 5 enhancer in P19 cells. ZNF423 overexpression and Ebf1 knockdown shows comparable reductions in enhancer activity (p,1027, Tukey HSD pair-wise comparisons to control after ANOVA). Combining ZNF423 overexpression and Ebf1 knockdown showed further reduction in.Es at ,2 mg per 16107cells. For ChIP-Seq, sheared chromatin was treated essentially as described [27] and converted to sequencing library for massively parallel sequencing on the Illumina GA-II platform. Sequencing was carried out in the UCSD BIOGEM Core facility. Analysis of resulting sequence reads performed in the Homer package [27] identified 13,765 Znf423 peaks at a calculated false discovery rate ,0.001. Tags were normalized to number of mapped reads for visualization in the UCSC Genome Browser as a custom track.Quantitative PCRPCR primers (Supplemental Table S1) were designed using Primer3 online tool [28]. Real-time PCR amplification was quantified by stimulated fluorescence of SYBR green dye on a Bio-Rad CFX-96 instrument. Relative quantification of ZNF423 among neuroblastoma lines compared expression in each sample to GAPDH as a conventional control by the DDCt method. For quantitative RT-PCR from mouse tissue and P19 cells, values were normalized to the geometric means of Gapdh, Pitpna, and Ppig reference genes and expressed as 22Ct(gene)/Ct(reference). Quantitative PCR from ChIP samples used either a pre-immune IgG mock ChIP or input fraction as indicated for relative quantification among samples.Materials and Methods AntibodiesZfp423 antibodies E20 and D16 were obtained from Santa Cruz Biotechnology (Figure 3A 16985061 ). Additional custom antisera were raised in rabbit against His-fusion protein expressing either residues 1?80 or 247?07 relative to human ZNF423 reference sequence NP_055884.2 and affinity-purified against the immunogen. ChIP experiments reported here used serum against 247?07 (Figure 3G ); serum against residues 1?80 performed less robustly in ChIP assays and was not considered further. EBF antibodies were a gift from Dr. Randall Reed (Figure 3A,B) or purchased from Santa Cruz Biotechnology (H300, Figure 3C ). SMAD antibodies A4 and H552 were obtained from Santa Cruz Biotechnology. Western blots were developed with infraredconjugated secondary antibodies (Rockland), detected on a LiCor Odyssey Imaging Station, and quantified in the ImageJWestern blotsHomogenized tissue, whole cell, or nuclear protein extracts were prepared in ice-cold RIPA buffer with protease inhibitor cocktail (Sigma), 10 mM DTT, 10 mM sodium orthovanadate, 8 M urea and treated with 100 U Benzonase nuclease (EMD) until minimize viscosity. Extracts were incubated in a sample buffer (50 mM Tris pH 6.8, 2 SDS, 0.1 Bromophenol blue 10 Glycerol, 33 mM DTT, 0.1 M b-mercaptoethanol, 8 M urea atZfp423 Binds Autoregulatory SitesFigure 5. Zfp423 overexpression represses intron 5 enhancer activity in P19 cells. (A) pGL4 reporter with the intron 5 enhancer was similarly active when co-transfected with shRNA directed against Zfp423 or a control. A similar plasmid with a region encompassing the intron 3 binding site had no activity above the pTAL minimal promoter. (B) Co-transfection with a plasmid expressing FLAG-tagged human ZNF423 reduced expression of the intron 5 reporter relative to a pcDNA vector control. This effect did not occur between paired samples with the Zfp423 consensus motifs mutated (intron5m). (C) An independent series of co-transfection assays indicates Ebf1-dependence of the intron 5 enhancer in P19 cells. ZNF423 overexpression and Ebf1 knockdown shows comparable reductions in enhancer activity (p,1027, Tukey HSD pair-wise comparisons to control after ANOVA). Combining ZNF423 overexpression and Ebf1 knockdown showed further reduction in.
