Figure 6 ER71 up-regulates VE-cadherin... | American Society of Hematology

Figure 6

$Figure 6. ER71 up-regulates VE-cadherin expression, which forms a VE-cadherin/ β-catenin/Flk-1 complex. (A) Cdh5 gene expression is shown in Flk-1+PDGFRα+ (DP) and Flk-1+PDGFRα− (SP) cell populations. (B-C) qRT-PCR analyses of Cdh5 gene expression in Er71+/+, Er71−/−, and iEr71 EBs (± DOX, with DOX during days 2-4) on day 4 (B; n = 4) and embryonic day 9.5 heart trunk region of Er71+/+ and Er71−/− embryos (n = 12; C). (D) Western blot analyses of VE-cadherin expression in day 4 Er71+/+, Er71−/−, or iEr71 EBs (± DOX, with DOX during days 2-4). (E) ER71 ChIP-Seq peaks in the Cdh5 gene region. The number of ChIP-Seq–aligned tags near the mouse Cdh5 gene locus is displayed in the UCSC genome browser. The location of the enriched ER71 ChIP-Seq peak relative to the transcription start site of the Cdh5 gene (−5.48 kb) is indicated with an arrow. (F) ChIP–qRT-PCR analysis for ER71 binding on the Cdh5 promoter region in iEr71 ES cells. Primers flanking an open reading frame–free intergenic region in mouse chromosome 6 were used as a negative control. Primers flanking the Flk-1 promoter region were used as a positive control. The relative enrichment of ER71 binding is represented as the -fold change. (G) Day 4 iEr71 EBs (± DOX) were subjected to immunoprecipitation using VE-cadherin (left), β-catenin (middle), or Flk1 Ab (right). Western blot analyses were performed using the Abs indicated on the left. DOX was added from day 2. (H-I) Day 4 iEr71 (H) and iβ-catenin-2A-Er71 (I) EBs (± DOX) were separated for cytosolic or nuclear fractions, and β-catenin levels were analyzed by Western blot. Cytosolic and nuclear signals were normalized to β-tubulin and lamin B, respectively. Data are presented as means ± SD of at least 4 independent experiments.$

ER71 up-regulates VE-cadherin expression, which forms a VE-cadherin/ β-catenin/Flk-1 complex. (A) Cdh5 gene expression is shown in Flk-1⁺PDGFRα⁺ (DP) and Flk-1⁺PDGFRα⁻ (SP) cell populations. (B-C) qRT-PCR analyses of Cdh5 gene expression in Er71^+/+, Er71^−/−, and iEr71 EBs (± DOX, with DOX during days 2-4) on day 4 (B; n = 4) and embryonic day 9.5 heart trunk region of Er71^+/+ and Er71^−/− embryos (n = 12; C). (D) Western blot analyses of VE-cadherin expression in day 4 Er71^+/+, Er71^−/−, or iEr71 EBs (± DOX, with DOX during days 2-4). (E) ER71 ChIP-Seq peaks in the Cdh5 gene region. The number of ChIP-Seq–aligned tags near the mouse Cdh5 gene locus is displayed in the UCSC genome browser. The location of the enriched ER71 ChIP-Seq peak relative to the transcription start site of the Cdh5 gene (−5.48 kb) is indicated with an arrow. (F) ChIP–qRT-PCR analysis for ER71 binding on the Cdh5 promoter region in iEr71 ES cells. Primers flanking an open reading frame–free intergenic region in mouse chromosome 6 were used as a negative control. Primers flanking the Flk-1 promoter region were used as a positive control. The relative enrichment of ER71 binding is represented as the -fold change. (G) Day 4 iEr71 EBs (± DOX) were subjected to immunoprecipitation using VE-cadherin (left), β-catenin (middle), or Flk1 Ab (right). Western blot analyses were performed using the Abs indicated on the left. DOX was added from day 2. (H-I) Day 4 iEr71 (H) and iβ-catenin-2A-Er71 (I) EBs (± DOX) were separated for cytosolic or nuclear fractions, and β-catenin levels were analyzed by Western blot. Cytosolic and nuclear signals were normalized to β-tubulin and lamin B, respectively. Data are presented as means ± SD of at least 4 independent experiments.

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