American Society of Hematology

Figure 3

$Figure 3. BAZF regulates polyubiquitination and degradation of CBF1. (A-B) Immunocytostaining of endogenous CBF1 in HUVECs with and without VEGF-A. Scale bar: 5 μm. (A) HUVECs were treated with VEGF-A for indicated hours and stained with anti-CBF1. (B) HUVECs were transfected with Mock, BAZF wild-type (WT), or BAZF-ΔZnF5. After transfection, the cells were stained with anti-CBF1. The images were acquired by ImageXpress Micro (Molecular Devices). The signal intensity of CBF1 staining in a hundred cells was measured by ImagePro Plus. Error bars show ± SD of independent triplicates (*P < .05, **P < .01). (C-D) Turnover of CBF1 in HUVECs. (C) After HUVEC were infected with AdBAZF or AdGFP, CHX was added to cell cultures at a concentration of 10μM. Cells were lysed at indicated hours post-CHX addition. Endogenous CBF1 in the chromatin-enriched nuclear matrix fraction was detected by Western blotting with an anti-CBF1 antibody. (D) HUVECs were treated with VEGF-A for indicated hours in the presence or absence of MG132. (E) Western blotting of CBF1 and BAZF in cytosol, nucleoplasm (soluble factions), and chromatin-enriched nuclear matrix (insoluble fraction) of AdBAZF or AdGFP infected HUVECs in the presence or absence of MG132. After infection and treatment of MG132, the cells were then harvested and fractionated to cytosol, nucleoplasm, and chromatin-enriched nuclear matrix fractions according to the protocol described in supplemental Methods. CBF1 and BAZF were detected by Western blotting with antibodies against each protein. Immunoprecipitation was performed using an anti-CBF1 antibody. Ubiquitinated CBF1 was detected by probing Western blots of the precipitates with an anti-ubiquitin antibody. Beta-tubulin, lamin A/C, and histone H3 were detected as fraction markers.$

BAZF regulates polyubiquitination and degradation of CBF1. (A-B) Immunocytostaining of endogenous CBF1 in HUVECs with and without VEGF-A. Scale bar: 5 μm. (A) HUVECs were treated with VEGF-A for indicated hours and stained with anti-CBF1. (B) HUVECs were transfected with Mock, BAZF wild-type (WT), or BAZF-ΔZnF5. After transfection, the cells were stained with anti-CBF1. The images were acquired by ImageXpress Micro (Molecular Devices). The signal intensity of CBF1 staining in a hundred cells was measured by ImagePro Plus. Error bars show ± SD of independent triplicates (*P < .05, **P < .01). (C-D) Turnover of CBF1 in HUVECs. (C) After HUVEC were infected with AdBAZF or AdGFP, CHX was added to cell cultures at a concentration of 10μM. Cells were lysed at indicated hours post-CHX addition. Endogenous CBF1 in the chromatin-enriched nuclear matrix fraction was detected by Western blotting with an anti-CBF1 antibody. (D) HUVECs were treated with VEGF-A for indicated hours in the presence or absence of MG132. (E) Western blotting of CBF1 and BAZF in cytosol, nucleoplasm (soluble factions), and chromatin-enriched nuclear matrix (insoluble fraction) of AdBAZF or AdGFP infected HUVECs in the presence or absence of MG132. After infection and treatment of MG132, the cells were then harvested and fractionated to cytosol, nucleoplasm, and chromatin-enriched nuclear matrix fractions according to the protocol described in supplemental Methods. CBF1 and BAZF were detected by Western blotting with antibodies against each protein. Immunoprecipitation was performed using an anti-CBF1 antibody. Ubiquitinated CBF1 was detected by probing Western blots of the precipitates with an anti-ubiquitin antibody. Beta-tubulin, lamin A/C, and histone H3 were detected as fraction markers.

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