Intron 5 site in two experiments among four replicates (Figure 3A,B). A single experiment further suggested possible Title Loaded From File binding to a predicted site in EBF3 gene. Interestingly, parallel ChIP-PCR experiments from the same lysates also detected EBF binding to several predicted sites, using an antibody with low discrimination among paralogous EBF members, though ChIP with several SMAD antibodies failed to detect binding under these conditions (Figure 3B). To extend these results, we examined whether the mouse homologs bound to the predicted intronic sites of Zfp423 and Ebf genes in P19 cells. Quantitative PCR detected modest but significant ChIP enrichment at the more 59 (and more deeply conserved) site in intron 3 (Figure 3C) and much stronger enrichment at the intron 5 site (Figure 3D) compared to IgG controls. These experiments did not confirm significant binding of either Zfp423 or Ebf to the tested sites in Ebf1 and Ebf3, nor did any site show a significant change in Zfp423 enrichment after activation of the SMAD pathway by BMP2 treatment (Figure 3E,F) in this cell type. To further confirm the binding of Zfp423 protein to the Zfp423 intron sites, we developed an independent affinitypurified serum against conserved epitopes near the aminoterminus and optimized ChIP conditions for Zfp423 in mouse P19 cells (Figure 3G,H). Affinity-purified serum provided substantially higher discrimination and higher percentage of input recovered compared to commercial preparations. Addition of 10 mM Zinc to each step of chromatin purification added a nominal increase in recovery. These experiments replicated the strong enrichment of the intron 5 site and provided additional evidence for binding at the intron 3 site, although the intron 5 site Biotin N-hydroxysuccinimide ester chemical information showed greater enrichment with both antibodies. Alignment of the predicted intron 5 binding site among orthologues showed that the consensus motif specially the core CCCnnGGG as strongly conserved among sequenced vertebrate genomes (Figure 3I). Western blots of total protein extracts from wild-type and Zfp423nur12 null mutant mouse forebrain and cerebellum showed specificity of both antibodies, with complete loss of Zfp423 in mutant brain (Figure 3J). The same membrane was probed sequentially with each antibody, imaged to ensure complete stripping in between, and imaged in alternate channels for the two species-specific secondary antibodies. As the custom serum gave stronger ChIP signal, its Western blot signal was imaged in the lower-sensitivity channel. Subsequent probing with antibodies against mouse b-actin and Gapdh confirmed similar levels of protein loaded across samples. To further test the specificity ofHuman IMR32 and Mouse P19 Cells Express ZfpBecause Zfp423 expression is developmentally dynamic and complex with respect to endogenous cell types [1,5], we briefly examined several cell lines that might be used as simplified model systems to assess direct binding to predicted targets sites with autoregulatory potential (Figure 2). Semi-quantitative RT-PCR from human cancer cell lines of neuroglial origin detected expression of ZNF423 and at least two of three EBF genes in both neuroblastoma (IMR32) and medulloblastoma (D238) derived cell lines (Figure 2A). By contrast, ZNF423 RNA was not detected in either of two glioblastoma lines (U87, U251). We further examined ZNF423 expression among four neuroblastoma cell lines by quantitative RT-PCR (qRT-PCR). IMR32 and SKN-SH each expressed high relative level of ZNF42.Intron 5 site in two experiments among four replicates (Figure 3A,B). A single experiment further suggested possible binding to a predicted site in EBF3 gene. Interestingly, parallel ChIP-PCR experiments from the same lysates also detected EBF binding to several predicted sites, using an antibody with low discrimination among paralogous EBF members, though ChIP with several SMAD antibodies failed to detect binding under these conditions (Figure 3B). To extend these results, we examined whether the mouse homologs bound to the predicted intronic sites of Zfp423 and Ebf genes in P19 cells. Quantitative PCR detected modest but significant ChIP enrichment at the more 59 (and more deeply conserved) site in intron 3 (Figure 3C) and much stronger enrichment at the intron 5 site (Figure 3D) compared to IgG controls. These experiments did not confirm significant binding of either Zfp423 or Ebf to the tested sites in Ebf1 and Ebf3, nor did any site show a significant change in Zfp423 enrichment after activation of the SMAD pathway by BMP2 treatment (Figure 3E,F) in this cell type. To further confirm the binding of Zfp423 protein to the Zfp423 intron sites, we developed an independent affinitypurified serum against conserved epitopes near the aminoterminus and optimized ChIP conditions for Zfp423 in mouse P19 cells (Figure 3G,H). Affinity-purified serum provided substantially higher discrimination and higher percentage of input recovered compared to commercial preparations. Addition of 10 mM Zinc to each step of chromatin purification added a nominal increase in recovery. These experiments replicated the strong enrichment of the intron 5 site and provided additional evidence for binding at the intron 3 site, although the intron 5 site showed greater enrichment with both antibodies. Alignment of the predicted intron 5 binding site among orthologues showed that the consensus motif specially the core CCCnnGGG as strongly conserved among sequenced vertebrate genomes (Figure 3I). Western blots of total protein extracts from wild-type and Zfp423nur12 null mutant mouse forebrain and cerebellum showed specificity of both antibodies, with complete loss of Zfp423 in mutant brain (Figure 3J). The same membrane was probed sequentially with each antibody, imaged to ensure complete stripping in between, and imaged in alternate channels for the two species-specific secondary antibodies. As the custom serum gave stronger ChIP signal, its Western blot signal was imaged in the lower-sensitivity channel. Subsequent probing with antibodies against mouse b-actin and Gapdh confirmed similar levels of protein loaded across samples. To further test the specificity ofHuman IMR32 and Mouse P19 Cells Express ZfpBecause Zfp423 expression is developmentally dynamic and complex with respect to endogenous cell types [1,5], we briefly examined several cell lines that might be used as simplified model systems to assess direct binding to predicted targets sites with autoregulatory potential (Figure 2). Semi-quantitative RT-PCR from human cancer cell lines of neuroglial origin detected expression of ZNF423 and at least two of three EBF genes in both neuroblastoma (IMR32) and medulloblastoma (D238) derived cell lines (Figure 2A). By contrast, ZNF423 RNA was not detected in either of two glioblastoma lines (U87, U251). We further examined ZNF423 expression among four neuroblastoma cell lines by quantitative RT-PCR (qRT-PCR). IMR32 and SKN-SH each expressed high relative level of ZNF42.
