Mitochondria isolation, BN PAGE, muscle protein lysates, western blots, GST affinity isolation, immunoprecipitation, antibodies

Mice were sacrificed and different muscles (hind limb, diaphragm) dissected and used for mitochondrial isolation or frozen in liquid nitrogen and used later for muscle lysates preparation and immunoblotting. For mitochondrial isolation and BN PAGE for the members of the TOMM complex we used previously reported protocols.^69,70 Pelleted mitochondria were solubilized in 50 µl ice-cold buffer (50 mM NaCl, 50 mM Imidazole-HCl, 1% Triton X-100, 5% [v:v] glycerol, 2 mM 6-aminohexanoic acid [Merck, 800145], 1 mM EDTA, pH 7.4 at 4°C) prior to addition of 5% (w:v) Coomassie Brilliant Blue G-250 (Serva Electrophoresis, 17524). BN PAGE was performed at 4°C using 4–13% gradient gels with 50 µg mitochondria loaded per lane. For SDS PAGE and immunoblotting, isolated mitochondria or muscles were homogenized in lysis buffer (10 mM HEPES, 400 mM NaCl, 10 mM KCl, 0.2 mM EDTA, 1% NP 40, 2 mM DTT, pH 7.9) with cOmplete protease inhibitors (Sigma-Aldrich Chemie, 04693116001) and phosphatase inhibitors (Sigma-Aldrich Chemie, P5726 and P0044). Skeletal muscle homogenates were sonicated for 10 sec and centrifuged at 16,100 g at 4°C for 5 min. Cleared lysate was used for immunoblotting experiments. Aliquots of mitochondrial or muscle lysates were solubilized in Laemmli buffer (150 mM Tris, pH 6.8, 6% SDS, 30% glycerol, 0.3% bromophenol blue, 3% ß-mercaptoethanol), boiled at 95°C, and loaded on 8% or 10% SDS-PAGE. Proteins were transferred to nitrocellulose membrane (Sigma Aldrich Chemie, Protran BA 85), blocked in 5% BSA (Carl Roth, 8076.4) or 5% nonfat dry milk (Heirler Cenovis, 3030) in PBS, 0.1% Tween20 (Carl Roth, 9127.1) for 1 h at room temperature.

For GST affinity-isolation assay, PINK1 and its epitopes were fused to GST and overexpressed in E. coli BL21-CodonPlus (DE3)-RP (Agilent Technologies, 230255). GST fusion proteins were harvested in 50 mM NaCl, 50 mM Tris, pH 7.6, 1 mM EDTA, 10% glycerol (Carl Roth, 3783), 2 mM DTT, 10 µg/ml leupeptin (AppliChem, A2183), 10 µg/ml aprotinin (Carl Roth, A162) and purified with GST beads (GE Healthcare Life Sciences, 17075601). Tom20, Tom22 (wild-type or mutant proteins), Tom40, and Tom70, all Tom proteins with T7 tag, were overexpressed in HEK293 cells and their interaction with recombinant GST fusion proteins was studied in 4.3 mM Na₂HPO₄, 1.47 mM KH₂PO₄, pH 7.0, 300 mM NaCl, 2.7 mM KCl, 1% Triton X-100.

Immunoprecipitation was done with specific antibodies for overexpressed proteins from cell lysates or endogenous proteins from diaphragm muscle lysates. Antibodies were incubated with lysates overnight at 4°C. The next day, protein A or protein G Sepharose beads (GE Healthcare Bio-Sciences AB, 10009441 and 17061801) were added and incubation was continued for another 2 h. Afterwards, beads were washed 3 times, boiled in Laemmli buffer and loaded on SDS PAGE gel.

Primary antibodies were incubated at 1:1,000 dilution or as mentioned. Following antibodies were purchased from Cell Signaling Technology: Histone H3 (4499; 1:5,000), MAP1LC3B (2775), MDH2 (8610). Additionally used antibodies: from Santa Cruz Biotechnology Inc.: TOMM20 (sc-11415; 1:3,000), TOMM40 (sc-11414), SLC25A31/ANT4 (detects in mouse also SLC25A4/ANT1, SLC25A5/ANT2) (sc-11433), PRKN/PARK2 (sc-32282); from Sigma-Aldrich Chemie; ACTN2 (A 7811; 1:10,000); from Abcam: PRKN/PARK2 (ab15954), SQSTM1 (ab56416; 1:5,000), MFN2 (ab56889; 1:5,000), VDAC1/2/3 (ab15895; 1:5,000), SDHA (ab14715; 1:20,000); from Abnova/Biozol: TOMM22 (H00056993-M01); from Novus Biologicals: PINK1 (BC100-494); from Proteintech: OPTN (10837-1-AP); from Merck: anti-phospho-S65 Ubiquitin (ABS1513); from Enzo Life Sciences: Mono- and polyubiquitinated conjugates (FK2); from Novagen: T7 (69522; 1.10,000); anti-p-S15-AA (generated in the lab of Dr. Michael Marber, UK); anti-CSNK2A1 (1:500) and anti-CSNK2A2 (1:100), both generated in the lab of Dr. Olaf-Georg Issinger (Odense, Denmark). Note, the VDAC antibody detects all three VDAC isoforms. VDAC1/2 show a very similar migration electrophoretic pattern and are indistinguishable in the immunoblot. VDAC3 migrates faster and separately detectable by with the VDAC antibody by immunoblot.⁷¹ Corresponding secondary antibodies conjugated with horseradish peroxidase (Cell Signaling Technology, 7074 and 7076; 1:3,000) were used for 2 h at room temperature. Protein bands were detected either by SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34095) or by homemade chemiluminescence reagent composed of 50 mg Luminol (Sigma Aldrich Chemie, A-4685) in 200 ml 0.1 M Tris, pH 8.6, combined with 11 mg para-hydroxy-cumarinic acid (Sigma Aldrich Chemie, C-9008) in 10 ml DMSO. For that, 3 ml of the first solution and 40 µl of the second solution were mixed with 3 ml of PBS and 1.2 μl 30% H₂O₂. Western Blot results were quantified by densitometric analysis using ImageJ software (NIH, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/). The background was subtracted with rolling ball radius 1,000 pixels and disabled smoothing option. Afterwards, protein bands of interest were labeled and measured. If not described specifically, values in mutant mice were expressed as relative values to control mice normalized to ACTN2 and set to 1.0.