Fig. 5: PRMT1 regulates fatty acid metabolism by methylating UBE2m at R169 in tubular epithelial cells.

A The scores of potential methylation sites of UBE2m predicted using the GPS-MSP tool. B MS identification of R169 mono-methylation of HA-UBE2m immunopurified by HA beads. C Sequence alignment of UBE2m protein from indicated species. D HA-UBE2m WT or different point mutants were co-transfected with FLAG-PRMT in HEK293T cells. The me1 level of UBE2m was measured by immunoblotting following immunopurification. E The me1 of immunopurified HA-UBE2m was measured using a site-specific antibody against R169 mono-methylation (meUBE2m (R169me1)). Antibody efficacy and specificity were examined by pre-incubating with the R169me1 peptide or the unmodified peptide prior to application. F Dot blot analysis of different amounts of R169me1 peptide or unmodified peptide by a site-specific antibody against meUBE2m (R169me1). G Endogenous UBE2m was immunoprecipitated in cells from the indicated groups. The R169me1 level of UBE2m was determined using immunoblotting. H The R169me1 level of UBE2m was determined using immunoblotting after UBE2m was immunoprecipitated in HK-2 cells with PRMT1 knockdown or overexpression. I Recombinant GST-PRMT1 and His-UBE2m proteins were incubated with or without SAM to detect the methylation of UBE2m mediated by PRMT1 in vitro. The reaction mixture was then subjected to CBB staining and immunoblotting with meUBE2m (R169me1) antibody. J Mitochondrial oxidative capacity was measured in real time after reintroduced UBE2m WT or RK treatment in UBE2m KO HK-2 cells; basal respiration, ATP production-coupled respiration, and maximal respiration were quantified. Significance was assessed using two-way ANOVA tests. Data are shown as mean ± SD.