Horseradish peroxidase conjugated anti rabbit or mouse antibodies

Total proteins were extracted in each group using RIPA buffer (CellNest, Minato, Tokoy, Japan) with Protease Inhibitor Cocktail Set III (1:1000, Millipore, Billerica, MA, USA). Protein concentration was measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s protocol. Protein samples were separated by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, blocked with 5% skim milk in Tris-buffered saline (TBS), and probed using various antibodies. The Western blots were visualized using ECL (Bio-rad) and exposed to Amersham™ Imager 600 (GE Healthcare Life Sciences, Uppsala, Sweden). The equivalence of protein loading was verified by probing for actin. The antibodies used were as follows: mouse anti-Mfn2 (1:500, Abcam); mouse anti-HO-1 (1:500, Abcam); rabbit anti-PGC1α (1:1000, Abcam); rabbit anti-iNOS (1:1000; Cell Signaling); rabbit anti-Nrf2 (1:1000, Abcam); rabbit anti-pink1 (1:500, Novus Biologicals, CO, USA); mouse anti-parkin (1:1000, Cell Signaling); mouse anti-β-actin (1:1000, Santa Cruz); horseradish peroxidase-conjugated anti-rabbit or -mouse antibodies (1:2500, Abcam).

Total proteins were extracted in each group using RIPA buffer (CellNest, Minato, Tokyo, Japan) with Protease Inhibitor Cocktail Set III (1:1000, Millipore, Billerica, MA, USA). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), in accordance with the manufacturer’s protocol. Protein samples were separated by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, blocked with 5% Difco^TM skim milk (BD Biosciences) in 1X Tris-buffered saline (TBS, Bio-rad) with 0.1% Tween 20 (Sigma), and probed using various antibodies. The Western blots were visualized using ECL (Bio-Rad) and exposed to the Amersham Imager 600 (GE Healthcare Life Sciences, Uppsala, Sweden). The equivalence of protein loading was verified by probing for actin. The antibodies used were as follows: rabbit anti-PTEN-induced putative kinase 1 (PINK1, 1:500, Novus Biologicals, CO, USA); mouse anti-parkin (1:1000, Cell Signaling); mouse anti-β-actin (1:1000, Santa Cruz); horseradish peroxidase-conjugated anti-rabbit or -mouse antibodies (1:2500, Abcam plc).