Fig. 5

CDC20 is capable of ubiquitinating CCDC69 in cardiomyocytes. a NRCMs were infected with adenovirus Ad-GFP and Ad-CDC20, treated with CHX and/or MG132, and harvested at the specified time points (n = 3); b CDC20 expression through western blotting after transfecting different concentrations of CDC20 plasmid into 293 T cells (n = 3); c Using Western blot detected the expression of CDC20 and CCDC69 by co-IP; d Protein lysates were immunoprecipitated with an anti-Flag antibody and analyzed by western blotting using Flag, Myc, and β-actin (n = 3); e Protein lysates were immunoprecipitated with an anti-Myc antibody and analyzed by western blotting using Flag, Myc, and β-actin (n = 3); f, g Lysates harvested from AC16 cells after transfecting myc-CCDC69 and Flag-CDC20, pretreated with MG132 for 6 h, and immunoprecipitated with anti-myc. The ubiquitin-conjugated CCDC69 was detected by Western blotting using anti-ubiquitin (Ub). Input shows the expression of the corresponding proteins in whole-cell lysates (n = 3); h CCDC69 and β-actin expression through western blotting (n = 3). i Transfection with myc-CCDC69, Flag-CDC20, and HA-UB, followed by immunoprecipitation with anti-myc. The ubiquitin-conjugated CCDC69 was detected by western blotting (n = 3); j Schematic diagram depicting the domain structure of CDC20 and a summary of in vitro binding experiments (n = 3); k Western blot showing the expression of CDC20 mutant proteins tagged with Flag (n = 3); l Western blot showing the expression of ubiquitin after transfecting CDC20 mutant plasmid (n = 3). NRCM: neonatal rat cardiomyocyte. Data presented as mean ± SD. Statistical analysis performed with one-way ANOVA or two-way ANOVA