Fig. 4: Parkin promotes the ubiquitination of MFF.

a The shRNA knockdown efficiency of Parkin and March5 was examined in SKOV-3 cells by real time PCR (n = 3 for each group). b SKOV-3 cells exogenously expressing Flag-MFF were knocked down with CPT1A, March5 or Parkin as indicated. Cells were harvested for immunoprecipitation with an anti-Flag antibody, followed by immunoblotting with an anti-Flag or anti-ubiquitin antibody, and the relative ubiquitin expression was normalized to Flag (IP) and then compared to control groups. c MFF was examined by WB in SKOV-3 cells with control, CPT1A knockdown or CPT1A and Parkin double knockdown. d SKOV-3 cells co-transfected with plasmids as indicated were treated with MG132 (10 μM) for 8 h. Cells were harvested for immunoprecipitation with an anti-Flag antibody. e SKOV-3 cells exogenously expressing Flag-MFF-WT were knocked down with CPT1A as indicated. Cells were harvested for immunoprecipitation with anti-Flag antibody, followed by immunoblotting with anti-Flag or anti-Parkin antibody. The relative Parkin expression was normalized to Flag (IP) and then compared to control groups. f SKOV-3 cells cotransfected with plasmids as indicated. Cells were harvested for immunoprecipitation with an anti-Flag antibody. Then, immunoblotting was performed with anti-Flag or anti-HA antibodies. The relative HA expression was normalized to Flag (IP) and then compared to control groups. g Representative confocal microscopy images of mitochondria in SKOV-3 cells with control, CPT1A knockdown or CPT1A and Parkin double knockdown (left). The proportion of cells with different mitochondrial morphologies was quantified (n = 3 samples, each sample contains 100 cellsright). Data represent the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, n.s. indicates no significant difference compared with the control groups. Scale bars, 10 μm.