Pink1

Vasicinone (purity > 98%) was purchased from Cayman Chemical (CAS-486-64-6, Ann Arbor, MI, USA). Paraquat, a Mitochondria Staining Kit (JC-1 stain) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The MitoSOXTM Red kit (M36008) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Dulbecco’s modified Eagle medium (DMEM:F12) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Primary antibodies against SOD-1, SOD-2, GST, GPx, TOM-20, VDAC-1, Parkin, PINK-1 and GAPDH were purchased from Santa Cruz Technology (Dallas, TX, USA), antibodies against DJ-1, α-synuclein, p-ULK, ATG7, ATG12 and LC3B were purchased from Cell Signaling Technology (Danvers, MA, USA) and an antibody against Nrf-2 was purchased from Abcam (Cambridge, MA, USA). All fluorescent secondary antibodies were purchased from Thermo Fisher Scientific in the USA.

The whole cell lysates, cytosol fraction lysates, or mitochondrial fraction lysates were prepared from TH1 cells and renal cortex tissue. Protein concentration was determined by employing the BCA method, and 30 μg of protein per sample was loaded for western blotting. Electrophoresis in 8–12% sodium dodecyl sulfate-polyacrylamide gel was followed by a transfer to polyvinylidene difluoride membranes. The blots were washed with TBST (10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20) and blocked with 5% skim milk for 1 h at room temperature. Subsequently, we incubated the blot with the appropriate primary antibodies: against PINK1 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-517353), SIAH3 (Novus, Littleton, CT, USA, NBP2-83524), LC3B (Novus, NB100-2220), P62/SQSTM1 (Novus, NBP1-48320), p-DRP1 (Ser637) (Cell Signaling Technology, #4867S), DRP1 (Santa Cruz Biotechnology, sc-271583), MFN1 (Santa Cruz Biotechnology, sc-166644), OPA1 (Novus, NBP1-71656), ubiquitin (Novus, NB300-130), VDAC1 (Novus, NB100-695), α-tubulin (Santa Cruz Biotechnology, sc-8035), and β-actin (Santa Cruz Biotechnology, sc-47778). Then, the membranes were washed again and incubated for detection with either goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies (Cell signaling, Danvers, MA, USA). The bands were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK).