Fig 1: High glucose induces SUMOylation of MuRF1. (A) WB of empty vector transfected C2C12 cells (mock) or cells after transfection with GFP, GFP-MuRF1, and GFP-MuRF1-K238R plasmids cultured in normal (N) and high glucose (H) medium for 6 h. Cell lysates were fractionated by SDS-PAGE and membranes probed with anti-SUMO2 antibodies to determine the conjugation level of SUMOylated protein. SUMOylated proteins decreased in cells cultured in high glucose medium and transfected with GFP-MuRF1-K238R. Anti-GFP antibodies were used to identify the GFP and GFP-MuRF1 protein levels and anti-GAPDH antibodies were used as protein loading control. (B) Quantification of SUMO2 conjugates from three independent experiments and normalized with the loading control GAPDH. ***P < 0.001, **P < 0.01. (C) Membranes in a were also probed with anti-PIAS4 and anti-UBC9 antibodies to detect the endogenous level of two SUMO-conjugating enzymes. (D) Quantification of three independent experiments normalized with the correspondent GAPDH signal as loading control. PIAS4 and UBC9 protein levels increased in cells cultured in high glucose medium. Gray bars indicate normal glucose (5.5 mM) and red bars indicate high glucose (25 mM). (E) Cellular ROS detection in C2C12 cells transfected with mock, GFP, GFP-MuRF1, and GFP-MuRF1-K238R plasmids and incubated for 6 h with fresh normal glucose (N) and high glucose (H) medium. ROS measurement was determined with fluorescence intensity as the ratio between excitation and emission values from 485/535 nm wavelength. The experiment was performed in triplicated from of three independent transfection experiments. The fluorescence detected in the non-transfected cells was removed from all the values. Cellular ROS activity was reduced in C2C12 cells transfected with GFP-MuRF1 and incubated in high glucose for 6 h before the assay. ***P < 0.001.