SUGCT converts itaconate to itaconyl-CoA. (A) Human SUGCT is homologous to the P aeruginosa itaconyl-CoA transferase (PaIct). Black and gray boxes highlight identical residues and conservative substitutions, respectively. (B) Discontinuous high-performance liquid chromatography (HPLC) assays showed that SUGCT generates itaconyl-CoA from succinyl-CoA and itaconate in vitro. (C) Immunoblotting of MEL mitochondria from cells treated with and without 2.5 mM itaconate and dimethyl sulfoxide (DMSO) for 72 hours. MEL mitochondrial lysates were probed with anti-ALAS2, anti-FECH, and anti-SUGCT primary antibodies. HPLC assays were performed on a C18 reverse-phase column with UV detection (mAU = milli-absorbance units at 254 nm). Optimal autoexposure within the linear dynamic range was performed for each immunoblot on the ChemiDoc imaging system (Bio-Rad). The HPLC chromatogram (B) and western blot (C) shown are representative of n = 2 biological replicates.