SDS-PAGE and BN-PAGE
One-dimensional 5–15% BN gradient and two-dimensional (2D) SDS gradient PAGE were done as described previously [54], [55]. Whole cell or tissue samples were used for SDS-PAGE and isolated mitochondria were used for BN-PAGE. SDS-PAGE with 10% NuPage gels (Invitrogen) and immunoblotting was performed as described previously [41]. Proteins were detected with the following antibodies: α-MNF1 (ATLAS antibodies or as previously described [25] for detection of UQCC2), Total OXPHOS Human WB Antibody Cocktail containing α-ATP5A1, α-UQCRC2, α-SDHB, α-COX2 and α-NDUFB8 (MitoSciences), α-NDUFA9 (as previously described [55]), α-UQCC (Atlas Antibodies), α-CBP (GenScript), α-ND1 (kindly provided by A Lombes [56]), α-cyclophilin B (Affinity Bioreagents), α-OXA1L (Central Animal Facility Nijmegen), α-SDHA, α-SDHB, α-COX1, α-UQCRC1, α-UQCRC2, α-UQCRFS1 and α-ATP5α (all MitoSciences), α-TOM20 (BD transduction laboratories), α-CK-B 21E10 (kindly provided by the Department of Cell Biology Nijmegen [57]), α-MRPL12 (Abcam), α-MRPS22 (Proteintech) and α-VDAC1 (Calbiochem). Secondary antibodies were goat α-mouse or swine α-rabbit IgG horseradish peroxidase (HRP, DakoCytomation), goat α-mouse or α-rabbit IgG HRP antibodies (Invitrogen). Quantification of western blots was performed by densitometry using ImageJ software or the Chemidoc XRS+ system (Biorad) software.