Figure 5.

WWP1 promotes KLF15-ubiquitination mediated degradation in hypoxia-induced cardiomyocytes. A NRCMs were infected with Adv-WWP1 or Adv-GFP for 48 h before the cells were treated with hypoxia and MG132 (5 μM) for 6 h, synchronously. Cellular proteins were isolated for immunoprecipitation with anti-KLF15 antibody followed by immunoblot with anti-Ub-K48 antibody. IgG as a negative control. n = 3. B Total expression level of KLF15 in NRCMs were examined by Western blots. Corresponding statistics of KLF15 were shown. n = 3. C H9C2 cells were transfected with Si-NC or Si-WWP1 for 48 h followed by hypoxic stimulation for 6 h. Total expression level of KLF15 were examined by Western blots. Corresponding statistics of KLF15 were shown. n = 3. D Expression levels of KLF15 in cytoplasm and nucleus in NRCMs were examined by Western blots. Corresponding statistics of KLF15 in cytoplasm and nucleus were shown. n = 3. E HEK293T cells transfected with vectors for His-Ub, GFP-KLF15, and either Flag-WWP1 or HA-C8886A mutant form of WWP1 were subjected to immunoprecipitation with the antibody against KLF15, and followed by immunoblot with anti-Ub-K48 antibody. IgG as a negative control. n = 3. F H9C2 cells were transfected with Flag-WWP1 plasmid or HA-C886A plasmid for 36 h before synchronous administration with hypoxia and MG132 (5 μM) for 6 h. Immunoprecipitation with the antibody against KLF15, and followed by immunoblot with anti-Ub-K48 antibody. IgG as a negative control. n = 3. G H9C2 cells were transfected with Flag-WWP1 plasmid or HA-C886A plasmid for 36 h before administration with hypoxia for 6 h. Total expression level of KLF15 were examined by Western blots. Corresponding statistics of KLF15 were shown. n = 3. The data are shown as the means ± SD. The data shown in B-D, and G were analysed by one-way ANOVA followed by Bonferroni post hoc test.