Fig. 5: A selective regulation of MacroD1 on mitochondrial complex I activity depends on the Macro domain.
A Blue native gel analysis of the expression of myocardial mitochondrial complexes in mice 18 hafter saline or LPS (10 mg/kg) injection. B–F Mitochondrial complexes activities in the hearts of mice 18 h after saline or LPS injection (n = 5 mice). G Schematic diagram of the sequence of MacroD1 and the MacroD1 mutant protein without the Macro domain (MacroD1 mut). H–K MitoSOX fluorescent staining (H, I; n = 5 cell samples from independent experiments), mitochondrial complex I activity (J; n = 4 cell samples from independent experiments), ATP levels (K; n = 5 cell samples from independent experiments) analysis in AC16 human cardiomyocytes transfected with plasmids of His-MacroD1, Flag-Ndufb9, or His-MacroD1 mut for 36 h and treated with and without LPS (1 μg/ml) for 18 h. L Venn diagram of MacroD1-binding proteins identified by antibody affinity proteomics, complex I-related proteins, and mono-ADP-ribosylated proteins immunocaptured by mono-ADP antibody. M Co-immunoprecipitation (Co-IP) for identifying endogenous interaction of MacroD1 and Ndufb9 in the hearts of mice. N Co-IP of MacroD1 and Ndufb9 in HEK293 cells transfected with His-MacroD1 and/or Flag-Ndufb9 plasmids for 48 hours. O His-MacroD1 or His-MacroD1 mutant with truncation of the Macro domain and Flag-Ndufb9 were co-expressed in HEK293 cells for Co-IP assay. Flox: MacroD1flox/flox controls; cKO: MacroD1flox/flox/αMHCMerCreMer mice. Data are shown as the mean ± SD. Data analysis was conducted utilizing one-way ANOVA with Tukey’s multiple comparison test. Source data are provided as a Source Data file.
