American Society of Hematology

Figure 4

$Figure 4. CUL3 mediates BAZF-induced degradation of CBF1. (A) Super-resolution imaging of colocalization of endogenous CUL3 and CBF1 on VEGF-A–induced ring structures. HUVEC were transfected with BAZF or control siRNA. After transfection and serum starvation, the cells were stimulated with VEGF-A for 6 hours. The cells were immunostained with anti-CUL3 (green) and anti-CBF1 (red) antibodies. The images were acquired by a structured illumination microscopy N-SIM (Nikon) to analyze colocalization of endogenous CUL3 and CBF1. Arrows: VEGF-A–induced ring structures including CUL3 and CBF1. Scale bar: 5 μm. (B-C) MRM analysis of CUL3 using hybrid triple quadrupole/linear ion trap mass spectrometer. (B) Representative MRM profiles of in-gel tryptic peptides from recombinant CUL3. Gel-separated CUL3 tryptic peptides in supplemental Figure 5 were subjected to MRM analysis. * indicates detected CUL3 ion peaks (enlarged view shown in the inset). MRM transitions for the targeted CUL3 peptides were as follows: NAIQEIQR (m/z 486.26→673.36, 786.45, and 299.17), EDGSEVGVGGAQVTGSNTR (m/z 607.29→635.31, and 734.38), and FLLESFNNDR (m/z 627.80→752.33, 881.37, and 994.46). (C) MRM analysis of endogenous CUL3 coimmunoprecipitated with CBF1. Tryptic digests of the immunoprecipitates were subjected to MRM analysis. * indicates Ion peaks assigned to the CUL3 peptide (an enlarged view is shown in the inset). CBF1 was also detected from immunoprecipitates by MRM analysis (data not shown). (D) Effect of CUL3 knockdown on polyubiquitination and degradation of CBF1 induced by AdBAZF infection. HUVECs were transfected with CUL3 siRNA or control siRNA. After transfection, HUVECs were infected with AdBAZF or AdGFP. And then, MG132 was added to the cell culture (50μM). The cells were harvested and fractionated into nucleoplasm and chromatin-enriched nuclear matrix fractions. Immunoprecipitation was performed using an anti-CBF1 antibody. Polyubiquitinated or nonpolyubiquitinated CBF1 was detected using anti-ubiquitin and anti-CBF1 antibodies. (E) Network formation of CUL3 knocked down HUVECs in the presence or absence of DAPT. HUVECs were transfected with CUL3 siRNA or control siRNA. The cells were seeded on the Matrigel with VEGF-A with or without DAPT. Twelve hours later, the images were acquired by a microscope. Scale bar: 20 μm.$

CUL3 mediates BAZF-induced degradation of CBF1. (A) Super-resolution imaging of colocalization of endogenous CUL3 and CBF1 on VEGF-A–induced ring structures. HUVEC were transfected with BAZF or control siRNA. After transfection and serum starvation, the cells were stimulated with VEGF-A for 6 hours. The cells were immunostained with anti-CUL3 (green) and anti-CBF1 (red) antibodies. The images were acquired by a structured illumination microscopy N-SIM (Nikon) to analyze colocalization of endogenous CUL3 and CBF1. Arrows: VEGF-A–induced ring structures including CUL3 and CBF1. Scale bar: 5 μm. (B-C) MRM analysis of CUL3 using hybrid triple quadrupole/linear ion trap mass spectrometer. (B) Representative MRM profiles of in-gel tryptic peptides from recombinant CUL3. Gel-separated CUL3 tryptic peptides in supplemental Figure 5 were subjected to MRM analysis. * indicates detected CUL3 ion peaks (enlarged view shown in the inset). MRM transitions for the targeted CUL3 peptides were as follows: NAIQEIQR (m/z 486.26→673.36, 786.45, and 299.17), EDGSEVGVGGAQVTGSNTR (m/z 607.29→635.31, and 734.38), and FLLESFNNDR (m/z 627.80→752.33, 881.37, and 994.46). (C) MRM analysis of endogenous CUL3 coimmunoprecipitated with CBF1. Tryptic digests of the immunoprecipitates were subjected to MRM analysis. * indicates Ion peaks assigned to the CUL3 peptide (an enlarged view is shown in the inset). CBF1 was also detected from immunoprecipitates by MRM analysis (data not shown). (D) Effect of CUL3 knockdown on polyubiquitination and degradation of CBF1 induced by AdBAZF infection. HUVECs were transfected with CUL3 siRNA or control siRNA. After transfection, HUVECs were infected with AdBAZF or AdGFP. And then, MG132 was added to the cell culture (50μM). The cells were harvested and fractionated into nucleoplasm and chromatin-enriched nuclear matrix fractions. Immunoprecipitation was performed using an anti-CBF1 antibody. Polyubiquitinated or nonpolyubiquitinated CBF1 was detected using anti-ubiquitin and anti-CBF1 antibodies. (E) Network formation of CUL3 knocked down HUVECs in the presence or absence of DAPT. HUVECs were transfected with CUL3 siRNA or control siRNA. The cells were seeded on the Matrigel with VEGF-A with or without DAPT. Twelve hours later, the images were acquired by a microscope. Scale bar: 20 μm.

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