Anti parkin antibody

The hippocampus of the brain was washed with PBS and then homogenized in 1 ml RIPA buffer with 100 μg/ml phenylmethylsulfonyl fluoride (Solarbio, Beijing, China) and further centrifugation at 12,000g for 20 min. The supernatant was collected, and the amount of protein was estimated by the BCA protein assay kit. Protein samples were loaded onto 10–12% sodium dodesyl sulfate (SDS) polyacrylamide gel, and then the proteins were transferred to polyethylene difluoride membranes (Millipore Corporation, USA). The membrane was blocked and subsequently incubated with anti-PINK1 antibody, anti-Parkin antibody, anti-superoxide dismutase 2 (SOD2) antibody and anti-NRF2 antibody (Abcam Inc, Cambridge, MA), anti-LC3B antibody (Genetex, Irvine, CA, USA), anti-dynaminrelated protein 1 (Drp1) antibody and anti-mitofusin 2 (Mfn2) antibody and anti-beta-actin antibody (Cell Signaling Technology, Inc., Beverly, MA, USA) for overnight at 4 °C. After primary antibody incubation was finished, the membranes were washed three times and incubated with HRP-conjugated secondary antibody. Secondary probes were detected by ECL Western blot detection reagents (GE Healthcare). The expression of protein was quantified using FluorChem FC2 software (Alpha Innotech Corporation).