Fig. 5: MRPL13 enhances ubiquitin-mediated degradation of SLC25A6.

A, B The protein and mRNA expression levels of SLC25A6 were measured by western blot and qRT-PCR in OVCAR-3 and ES-2 cells with MRPL13 knockdown or overexpression, respectively. β-Actin was used as an internal control. C Stability analysis of SLC25A6 protein half-life was validated by western blot in MRPL13-knockdown OVCAR-3 cells after treatment with 20 μg/mL CHX for the indicated times. D Stability analysis of SLC25A6 protein half-life was validated by western blot in OVCAR-3 cells after treatment with 20 μg/mL CHX and either 10 μM MG132 or 400 nM BafA1 for the indicated times. E, F SLC25A6 protein half-life plots were derived by quantifying the relative levels of SLC25A6 to β-actin protein, based on band intensity. G, H Ubiquitination assay of SLC25A6 in OVCAR-3 and ES-2 cells with MRPL13 overexpression treated with 10 μM MG132 for 6 h. Cell lysates were immunoprecipitated in the presence of anti-SLC25A6 antibodies. Immunoblotting was performed using anti-Ubiquitin and anti-SLC25A6 antibodies. I Ubiquitination assay of SLC25A6 in HEK293T cells co-transfected with HA-Ub, Myc-SLC25A6 or Flag-MRPL13 and treated with 10 μM MG-132 for 6 h. Cell lysates were immunoprecipitated in the presence of anti-Myc antibodies. Immunoblotting was performed using anti-HA and anti-Myc antibodies. J Ubiquitination assay of SLC25A6 in HEK293T cells co-transfected with Myc-SLC25A6, Flag-MRPL13 or the HA-tagged K6-, K11-, K27-, K29-, K33-, K48-, or K63-only ubiquitin mutant (for example, K6 indicates that all lysines with the exception of K6 were mutated to arginine) and treated with 10 μM MG-132 for 6 h. Cell lysates were immunoprecipitated in the presence of anti-Myc antibodies. Immunoblotting was performed using anti-HA and anti-Myc antibodies. All assays were performed in three independent experiments. Data are presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.