Ab15494

Isolation of mitochondria from mouse heart, skeletal muscle and brain was performed by differential centrifugation^{67 (link)}. Total extracts were prepared from mouse ventral midbrain isolated from coronal brain cryosections and from control and patient fibroblasts using RIPA buffer supplemented with protease inhibitors (Complete, Roche). Five micrograms of isolated mitochondria or twenty micrograms of total protein extracts were resuspended in Laemmli buffer, run on 12% SDS–polyacrylamide gel electrophoresis (Invitrogen) and then transferred onto nitrocellulose membrane using iBlot2 system (Invitrogen). Immunodetection was performed according to standard techniques using enhanced chemiluminescence Immun-Star HRP Luminol/Enhancer (Bio-Rad). The following antibodies were used: PORIN (ab14734, Abcam), PARKIN (ab15494, Abcam), TH (MAB318, Chemicon), HSC70 (sc-7298, Santa Cruz) and OXPHOS Rodent Antibody Cocktail (ab110413, Abcam) for NDUFB8 (CI), SDHB (CII), UQCRC2 (CIII), MTCO1 (CIV) ATP5A (CV).
All blots were processed in parallel and derive from the same experiments. Quantification of Western blot signals was performed by densitometry using software FIJI (ImageJ2; Version 2.3.0/1.53f).

The total proteins in the testis tissues were separated using a Total Protein Extraction Kit (BC3711, Solarbio). The protein concentration was measured using a Protein Assay kit (Beyotime). Next, the protein samples were separated by 10% SDS-PAGE and electrically transferred to PVDF membranes (Millipore, MA, USA). After sealing with 3% bovine serum albumin at room temperature for 1 h, the membranes were hatched with the primary antibodies (rabbit anti-prohibitin (PHB), ab28172, 1:1000; rabbit anti-Beclin-1, ab62557, 1:2000; rabbit anti-LC3II/I, ab128025, 1:1,000; rabbit anti-p62, ab56416, 1:1,000; rabbit anti-SCF, ab64677, 1:1000; rabbit anti-Parkin, ab15494, 1:1,000; rabbit anti-LKB1, ab199970, 1:1,000; rabbit anti-AMPKα, ab131512, 1:500; rabbit anti-phosphorylated AMPKα (p-AMPKα), ab133448, 1:10000; rabbit anti-ULK1, ab167139, 1:1000; rabbit anti-p-ULK1, ab203207, 1:1000; rabbit anti-β-actin, ab8227, 1:5000; Abcam, Cambridge, UK) overnight at 4 °C. The membranes were incubated with goat-anti-rabbit IgG (H + L)-HRP (1:10000, ab6721, Abcam) for 1 h at room temperature and then rinsed thrice with TBST thrice. Protein bands were visualized using an Electrochemilluminescence (ECL) chemiluminescence kit (WBULS0500; EMD Millipore), and band intensity was analyzed with Image-Pro Plus 6.0.