Research Paper Volume 12, Issue 7 pp 5716—5732

Ube2s-stabilized β-catenin protects against myocardial ischemia/reperfusion injury by activating HIF-1α signaling

Figure 5. Ube2s stabilizes β-Catenin after MI/R injury. (A) C57BL/6 mice were intra-myocardially infected with lentivirus expressing vector control or Ube2s 48 h prior to MI/R surgery. Following 24 h of reperfusion, the protein level of β-Catenin in the heart was analyzed by Western blotting. Samples from sham group were used as controls. Each group includes 8 mice. β-Actin was used as a loading control. The representative band images (left) and relative band intensity analysis (right) are presented. Data are mean ± SD. Data were compared using Student’s t-test. **, P < 0.01. (B) C57BL/6 mice were intra-myocardially transfected with control siRNA (siCtrl) or Ube2s siRNA (siUbe2s) 48 h prior to MI/R surgery. Following 24 h of reperfusion, the protein level of β-Catenin in the heart was analyzed by Western blotting. Samples from sham group were used as controls. Each group includes 8 mice. β-Actin was used as a loading control. The representative band images (left) and relative band intensity analysis (right) are presented. Data are mean ± SD. Data were compared using Student’s t-test. **, P < 0.01. (C) The cardiomyocytes overexpressing empty vector or Ube2s were treated with cycloheximide (CHX) for increasing time periods as indicated. The protein expression of β-Catenin and Ube2s was determined by Western blotting analysis. β-Actin was used as a loading control. The representative band images (left) and relative band intensity analysis of β-Catenin (right) are presented. The half-life is depicted by dot line. Data are mean ± SD. Data were compared using Student’s t-test. **, P < 0.01. (D) The lysates of cardiomyocytes stably overexpressing empty vector or Ube2s were immunoprecipitated (IP) with β-Catenin antibody. The IP products were further analyzed by Western blotting to detect ubiquitin expression. The expression of β-Catenin and Ube2s in the input fraction is presented below. (E) The lysates of heart tissues from mice, intra-myocardially infected with lentivirus expressing vector control or Ube2s 48 h prior to MI/R surgery, were immunoprecipitated (IP) with β-Catenin antibody. The IP products were further analyzed by Western blotting to detect ubiquitin expression. The expression of β-Catenin and Ube2s in the input fraction is presented below.