Fig. 3: CPT1A inhibits the ubiquitination of MFF to stabilize MFF.

a, b SKOV3 cells exogenously expressing Flag-MFF-WT were treated with 10 μM MG132 (a) or 5 μM chloroquine (CQ, b) for up to 12 h, and the expression of endogenous MFF, Flag-MFF and CPT1A was assessed by immunoblotting. Relative expression of Flag was normalized to β-tubulin and then compared to 0 h. c SKOV-3 cells cotransfected with plasmids as indicated were treated with MG132 (10 μM) for 8 h. Cells were harvested for immunoprecipitation with anti-Flag antibody. Bands are derived from the different gels. d 293TN cells cotransfected with plasmids as indicated were treated with MG132 (10 μM) for 8 h. Cells were harvested for immunoprecipitation with anti-Flag antibody. The relative HA expression was normalized to Flag (IP) and then compared to control groups. e Mass spectrometry analysis of MFF ubiquitinated peptides. The red arrow points to the parental ions of interest subjected to tandem MS analysis, where the ubiquitination site occurs. f Multi-species conservation analysis of ubiquitination modified sequences. g 293TN cells transfected with Flag-MFF-WT or Flag-MFF-K315R (Flag-K315R) plasmids were treated with MG132 (10 μM) for 8 h. Cells were harvested for immunoprecipitation with anti-Flag antibody and immunoblotting with anti-Flag and anti-ubiquitin antibodies. The relative ubiquitination of MFF was determined by normalizing ubiquitin to Flag (IP). h 293TN cells transfected with Flag-MFF-WT or Flag-MFF-K315R (Flag-K315R) were treated with 10 μM cycloheximide (CHX) for up to 12 h. Cells were harvested for immunoblotting with anti-Flag and anti-MFF antibodies. Beta-Tubulin was used as a loading control. Bands are derived from the different gels. Densitometric analysis of the Flag immunoblot band was performed with ImageJ (n = 3 for each group). 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.