Complete protease inhibitor cocktail mix

Homogenized rat liver was lysed in 200 μl RIPA lysis buffer (Beyotime, P0013B) with 1% phenylmethyl sulfonylfluoride and 4% complete protease inhibitor cocktail mix (Roche, Mannheim, Germany). Extracts were centrifuged at 14,000 g for 15 min at 4°C. Eighty micrograms of total protein were used for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, followed by transferring blotting to nitrocellulose membrane (Millipore Corp., Billerica, MA, USA). Membranes were then blocked with 5% nonfat dried milk in PBS for 1 hr with gentle shaking. Membranes were incubated first with anti‐Janus kinase 2(JAK2), anti‐signal transducer and activator of transcription 3 (STAT3) and their phosphorylated species, anti‐cytochrome P450 2E1(CYP2E1), anti‐Nuclear factor erythroid 2 related factor 2 (Nrf2), anti‐heme oxygenase‐1(HO‐1), anti‐NAD(P)H: quinone oxidoreductase 1 (NQO‐1), and anti‐β‐actin antibody [Cell Signaling Technology] were incubated overnight at 4°C, in 1% BSA in PBS overnight at 4°C with shaking, washed and incubated with secondary antibodies for 2 hr at room temperature. Finally, the samples were visualized by enhanced chemi‐luminescence. After scanning, band density was analyzed using Image J 1.33 software (National Institutes of Health, Bethesda, MD, USA).

Directly after image acquisition at the microscope, cells were washed twice with sterile PBS and then lysed on ice for 10 min in lysis buffer as previously described^{17 (link)} or RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 0.5% DOC, 0.1% SDS, complete protease inhibitor cocktail mix, Roche). Lysates were centrifuged at 13 000 r.p.m. for 10 min at 4 °C. Supernatants were collected and protein concentration was determined by the Bradford method. Samples were stored at −80 °C until used. 10 to 30 μg of protein per slot were separated on a 12% SDS polyacrylamide gel and transferred to a polvinylidene difluoride membrane. Membranes were incubated for 1 h in TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween, pH 7.5) containing 5% milk to saturate all non-specific binding sites. Incubation with primary antibodies was overnight at 4 °C in 1% milk+TBST (1 : 1500 goat anti-DJ-1, Santa Cruz Biotechnology, Dallas, TX, USA; #Sc-27006; 1 : 3000 mouse anti-α-Synuclein, BD #610787; 1 : 5000 mouse anti-β-actin, Sigma-Aldrich, St. Louis, MO, USA; #A5441). Blots were developed using horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-goat 1 : 10 000 Jackson Immunoresearch, West Grove, PA, USA; #705-035-003, anti-mouse 1 : 10 000 Sigma #50185-ILM-F) and the Immobilon chemiluminescent HRP substrate (Millipore, Billerica, MA, USA).