Fig 1: MA-5 accelerated ATP production with the ATP synthase dimer resulting in supercomplex formation.(a) In gel ATP synthase activity. Native gel of whole, solubilized bovine heart mitochondria with two different concentrations of digitonin (0.5% or 1.0%) stained for ATP synthesis activity. The bands corresponding to the ATP synthase monomer and dimer were indicated by arrowheads. Note that MA-5 increased the intensity of ATPase dimer and multimer formation.(b) Western blotting analysis of Blue-Native gels. The same amounts of isolated mitochondria were treated with three different concentrations of digitonin (0.25%, 0.5% and 1%) and treated with DMSO or MA-5. The resultant protein was electrophoresed and transferred on a membrane. Antibody against ATP5A was used.(c) Comparison with the western blotting densitometric analysis on BN-PAGE representing 0.25% digitonin panle. The monomer (left panel) and dimer (right panel) bands were standardized by VDAC1, which is the loading control on SDS-PAGE.(d) Western blotting analysis of Blue-Native gel treated with DMSO and MA-5 with ATP5A, ATP5B, ATP5I (e), ATP5L (g) and mitofilin/Mic60 antibodies.(e) The LC/MS analysis of the supercomplex components enhanced by MA-5 on BN-PAGE.(f) Putative mitofilin/Mic60-binding proteins identified by affinity chromatography and mass spectrometry (Kanno et al., 2007).(g) Elution profile of MA-5 treatment or non-treatment IMMT and ATP5A on size-exclusion chromatography. MA-5 treatment (10 µM, 1 h) and non-treatment HEC293T cell extracts were separated on a Superose™ 6 (GE Healthcare). Proteins of each fraction were then separated by SDS-PAGE and analyzed by western blotting with an anti-IMMT antibody and anti-ATP5A antibody. After MA-5 treatment, the fractions containing ATP5A (upper) and mitofilin/Mic60 (lower) were shifted to the higher fraction size.