Figure 3. MTND1 mutations lead to a loss of immunoreactive CI subunits and impair CI assembly.

(A) SDS/PAGE and Western blot analysis of muscle lysates (20 μg) from two controls (Co1, Co2) and two patients (P1, m.3365T>C mutation; P2, m.4175G>A mutation). Membranes were probed with antibodies directed against mitochondrial respiratory chain subunits and porin as a loading control. (B) Relative protein expression levels. Band intensity of indicated protein is normalized to band intensity of the loading control porin. Shown are mean values of three independent experiments ± S.D. *P<0.05 relative to controls; ns, not significant. (C) BN/PAGE of dodecyl maltoside (DDM)-solubilized muscle samples (75 μg) from two controls (Co1, Co2) and both patients with MTND1 mutations (P1, P2) followed by Western blot analysis. Membranes were probed with anti-NDUFA9 for CI, SDHA for CII, CORE2 for CIII₂ and COX4 for CIV. Blots are representative of two independent experiments. (D) In-gel enzyme activity (IGA) assay of respiratory chain complexes. BN/PAGE was performed on DDM-solubilized muscle samples (150 μg) from two controls and both patients. Enzyme activities for CI, CIV and CII were examined. (E) BN/PAGE of digitonin-solubilized muscle samples (100 μg) from two controls (Co1, Co2) and both patients (P1, P2) followed by Western blot analysis. Membranes were probed with anti-NDUFA9 and with anti-CORE2. SC1, CIII₂ + CIV; SC2, CI + CIII₂; SC3, CI + CIII₂ + CIV. Blots are representative of two independent experiments. (F) IGA assay of mitochondrial super-complexes. BN/PAGE was performed on digitonin-solubilized muscle samples (200 μg) from two controls and the two patients with MTND1 mutations (P1, P2). Enzyme activities for CI and CII were examined. SC1, CIII₂ + CIV; SC2, CI + CIII₂; SC3, CI + CIII₂ + CIV.