Pink1

Equal amounts of proteins were applied to SDS-PAGE and electroblotted on a PVDF membrane. Immunoblotting analysis was performed using a chemiluminescence detection kit. The relative levels of immunoreactivity were determined by densitometry using the free software ImageJ (National Institutes of Health, Bethesda, MD, USA).
Primary antibodies used were as folllows: Actin (1 : 20 000; Sigma-Aldrich, St. Louis, MO, USA; A5060), glyceraldehyde 3-phosphate dehydrogenase (1 : 10 000; Calbiochem, Darmstadt, Germany; CB1001), Tom20 (1 : 1000; Santa Cruz, sc-11415), MnSOD (1 : 2000; Enzo Life Sciences, Lausen, Switzerland; ADI-SOD-110), DLP1 (Drp1, 1 : 2000; BD Transduction Laboratories, San Jose, CA, USA; 611112), phospho-Drp1 Ser637 (1 : 1000; Cell Signaling, Danvers, MA, USA; 4867), Mfn1 (1 : 1000; Santa Cruz, sc-50330), parkin (1 : 500; Santa Cruz, sc-32282), LC3 (1 : 250; NanoTools, Teningen, Germany; 0231-100/LC3-5F10), p62/SQSTM1 (1 : 1000; MBL, Woburn, MA, USA; PM045), PINK1 (1 : 1000; Sigma-Aldrich, P0051), DSCR1 (RCAN1, 1 : 1000; Santa Cruz, sc-66864), cytochrome c (1 : 1000; BD Pharmingen, San Jose, CA, USA; 556433), multi ubiquitin (1 : 1000; MBL, D058-3).
Secondary antibodies used were as folllows: goat anti-mouse IgG (1 : 3000; Bio-Rad 170–6516), goat anti-rabbit IgG (1 : 3000; Bio-Rad 170–6515).

Cellular protein was extracted using RIPA buffer (1% Triton X-100, 50 mM KCl, 25 mM Hepes, pH 7.8, 10 µg/ml leupeptin, 20 µg/ml aprotinin, 125 µM dithiothreitol, 1 mM Phenylmethanesulfonyl fluoride, and 1 mM sodium orthovanadate), supplemented with protease inhibitors (Cat No. P8340, Sigma-Aldrich, USA) followed by centrifugation at 10,000 rpm for 20 min. The protein concentration in the supernatant samples was determined by Qubit protein assay kit (Molecular Probes, Invitrogen, CA, USA). Cell lysates were resolved on standard SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Millipore, Bedford, MA, USA) using a semi-dry transfer system (Amersham Biosciences, GE Healthcare, USA). The blots were incubated overnight with primary antibodies against PINK1 (1 µg/ml; Sigma-Aldrich, USA), PARKIN (1 µg/ml), MFN2 (2.51 µg/ml), NIX (0.75 µg/ml), LC3-II (1.5 µg/ml), and LAMP-2 (2 µg/ml), obtained from Abcam (Cambridge, MA, USA), followed by probing with their respective HRP-conjugated secondary antibodies. Blots were scanned using an enhanced chemiluminescence system (Fluorchem M, Protein simple), and the band intensity of target proteins was normalized to β-actin by Image J software (1.47 v).

Western blotting was performed as previously described^{17 (link)} using rabbit primary antibodies to SIRT3 (#5490, Cell Signaling Technology, Danvers, MA, USA), OPA1 (#80471, Cell Signaling Technology), cleaved caspase-3 (Asp175) (Cell Signaling Technology) and PINK1 (Sigma-Aldrich) diluted 1:1000. Mouse monoclonal primary antibodies to α-tubulin (DM1A) (Cell Signaling Technology) and β-actin (A5441, clone AC-15) (Sigma-Aldrich) were diluted, respectively, 1:1000 and 1:5000 before use. Horseradish peroxidase conjugated anti-mouse and anti-rabbit IgG (Cell Signaling Technology) secondary antibodies were used at 1:2,000 dilution. A semi quantitative measurement of band intensity was performed using the Carestream Molecular Imaging Software (Carestream Molecular Imaging, New Haven, CT, USA) and Fiji^{36 (link)}.

PGAM5 antibody was made against the peptide, CGSLEKDRTLTPLGR by Genscript (NJ, USA). Commercial antibodies were RIP1 (BD Bioscience); RIP3 (eBioscience); PINK1 and PARL (Sigma); and COXIV, VDAC, Actin, HSP60, HMGB1 and tubulin antibodies (Cell Signaling Technology).