Secondary hrp conjugates

Cells were prepared as described in ref. ^{44 (link)}. Briefly, whole-cell pellets were solubilized in phosphate buffered saline, 1% dodecyl-maltoside (DDM), 1 mM PMSF (phenylmethylsulfonyl fluoride), and complete protease inhibitor (Thermo Fisher). Protein concentrations were measured by the Bradford assay (BioRad). Equal amounts of proteins were separated by Tris-Glycine SDS-PAGE. Membranes were blocked in TBST with 1% milk at room temperature for 1 h then primary antibodies (Proteintech Group: MTO1 (15650-1AP, 1:5000); TRMT61B (26009-1AP, 1:2000); MT-CYB (55090-1-AP, 1:1000); MT-ATP6 (55313-1-AP, 1:2000); MRPL11 (15543-1-AP, 1:20000); MRPL45 (15682-1-AP, 1:5000); MRPS27 (17280-1-AP, 1:5000). Abcam: MT-CO1 (1D6E1A8, 1:1000); SDHA (2E3GC12FB2AE2, 1:10000). Santa Cruz: TOM40 (sc-11414, 1:2000)) were incubated overnight at + 4 °C in 5% BSA/TBST and detected the following day with secondary HRP conjugates (Jackson ImmunoResearch) using ECL with film. Representative data of independent experiments were cropped in Photoshop with only linear corrections applied.

The cDNA for wild type and mutant MPV17 with a C-terminal HA-epitope tag was cloned into a Gateway (Invitrogen, California, USA) converted retroviral expression vector pBABE-puro. Retrovirus was generated by transient transfection into the Phoenix amphotropic packaging line and transduced into mouse embryonic fibroblasts (MEF). Following antibiotic selection, cells were directly used in experiments. MEFs were solubilized in phosphate buffered saline, 1% dodecyl-maltoside, 1 mM PMSF (phenylmethylsulfonyl fluoride). Protein concentrations were measured by the Bradford assay (BioRAD, California, USA). Equal amounts of proteins were separated by Tris-Glycine SDS-PAGE and transferred to nitrocellulose by semi-dry transfer. Primary antibodies (anti-HA, Sigma-Aldrich, Missouri, USA; anti-Tom40, Santa Cruz Biotech, Texas, USA) were incubated overnight at +4°C and detected the following day with secondary HRP conjugates (Jackson ImmunoResearch, Pennsylvania, USA) using ECL with film.

Cells were solubilized in phosphate buffered saline, 1% dodecyl-maltoside (DDM), 1 mm phenylmethylsulfonyl fluoride (PMSF) and complete protease inhibitor (Thermo Fisher). Protein concentrations were measured by the Bradford assay (BioRad). Equal amounts of proteins were separated by Tris-Glycine SDS-PAGE and were transferred to nitrocellulose membrane by semi-dry transfer. Membranes were blocked in TBST with 1% milk at room temperature for 1 h, followed by incubation with primary antibodies overnight at 4°C in 5% BSA/TBST. Signals were detected the following day with secondary HRP conjugates (Jackson ImmunoResearch) using ECL with X-ray film and iBright Imaging System (Thermo Fisher). Primary antibodies from Proteintech Group: AFG3L2 (14631-1-AP, 1:5000), uL11m (15543-1-AP, 1:20 000), mS27 (17280-1-AP, 1:5000) and OXA1L (21055-1-AP, 1:5000). Additional primary antibodies used were MTCO1(1D6E1A8, 1:500) (Abcam/Mitosciences) and TOM40 (sc-11 414, 1:5000) (Santa Cruz). Representative data of independent experiments were cropped in Adobe Photoshop with only linear corrections to brightness applied.