BN-PAGE, SDS-PAGE, and Complementation Studies
(A) Mitochondria isolated from cultured skin fibroblasts from subjects 1–3 and age-matched control subjects were solubilized in n-dodecyl β-d-maltoside (DDM) and subjected to BN-PAGE and immunoblotting analysis using antibodies directed to various OXPHOS complexes as indicated. The blot probed with an antibody raised against NDUFB8 revealed the presence of additional, partially assembled complex I intermediates in the samples from subjects 1–3 (indicated by an asterisk) but not in control samples.
(B) Whole-fibroblast cell lysates from subjects 1–3 and age-matched control subjects were analyzed by SDS-PAGE. Immunoblotting was performed with antibodies against complex I subunits or control proteins (SDHA and porin) as indicated. Complex I structural subunits are color coded according to their corresponding complex I modules, as illustrated in the complex I pictogram.
(C) Wild-type NDUFA6 cDNA was generated and introduced into control and subject cell lines via retroviral expression. Whole-cell lysates were solubilized in 1% Triton X-100 (immunoblotting) before BN-PAGE analysis. Immunoblotting using antibodies against the complex I subunit NDUFA9 (top) and the complex II subunit SDHA (bottom) as a loading control revealed less complex I in subject cell lines than in control cell lines. After transfection with NDUFA6, complex I levels were restored.
(D) In-gel activity analysis was performed according to Zerbetto et al.22 with mitochondria isolated from the fibroblasts of subjects 1 (S1) and 3 (S3) with (+) and without (−) lentiviral transduction with NDUFA6 cDNA and an aged-matched control subject; enriched mitochondria were solubilized in 1% digitonin for BN-PAGE analysis, which demonstrated restoration of complex I activity in subjects 1 and 3 after the introduction of NDUFA6 (top). The gel was then stained with colloidal Coomassie according to Neuhoff et al.23 as a loading control (bottom). All antibodies used are documented in Table S1.