Heme oxygenase 1 ho 1

Western blot analyses were performed as previously described (Wang et al., 2011 (link)). Animals were anesthetized and sacrificed by cervical dislocation. The brain tissue was immediately harvested and stored at −80°C. The tissue dissected from mouse brain was homogenized in RIPA buffer. After centrifuging at 12,000 rpm for 5 min, supernatant was collected as the total cell lysate. Protein concentration was measured using the BCA Protein Assay Kit from Thermo Fisher Scientific. Supernatant was separated on 4%–15% polyacrylamide Tris-HCl gradient gels (BioRad, Hercules, CA, USA) and transferred to PVDF membranes. The integrated optic density of the immunoreactive band was quantified with NIH ImageJ software. The primary antibodies used were: rabbit polyclonal superoxide dismutase 2 (SOD2; 1:2000; EMD Millipore), rabbit polyclonal glutathione peroxidase 1 (GPx1; 1:1000; Abcam, Cambridge, MA, USA), goat polyclonal NAD(P)H:Quinone Oxidoreductase 1 (NQO1; NQP1; 1:1000; Abacam), rabbit polyclonal heme oxygenase 1 (HO1; 1:1000; Enzo Life Sciences, Farmingdale, NY, USA) and mouse anti-actin (1:5000, EMD Millipore).

Western blotting for Glutathione Peroxidase 1 (Gpx1) (ABCAM, Billerica, MA), GSTπ (MBL International, Woburn, MA), GSTμ (ABCAM, Cambridge, MA), Heme Oxygenase 1 (HO-1) Enzo Life Sciences, Farmingdale, NY, GCLC (Novus Biologicals, Littleton, CO), P62 (SQSTM1, Sequestosome 1) mouse monoclonal (ABCAM), LC3B (Novus Biologicals), anti-GAPDH (Millipore, Billerica, MA) was performed from 10 μg of liver extracts as previously described [10 (link), 26 (link)–28 (link)]. Quantification of expression of each protein was performed using ImageJ (NIH) and normalized to overall GAPDH expression.

Total protein extracts were prepared by homogenizing samples in lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 2 mmol/L EDTA, 2 mmol/L EGTA, 0.2% Triton X-100, 0.3% NP-40, 0.1 mmol/L PMSF, 25 mmol/L NaF). For nuclear extracts, the NE-PER^TM nuclear and cytoplasmic extraction kit (Pierce, Thermofisher Scientific) was used according to the manufacturer’s instructions. Proteins were separated by SDS-PAGE under reducing conditions and then blotted onto nitrocellulose membranes. Membranes were blocked with 5% defatted milk in TBS-T (0.05 mol/L Tris, 0.15 mol/L NaCl, 0.05% Tween 20, pH 7.8). Thereafter, membranes were probed overnight at 4 °C with primary antibodies in the same blocking solution or 5% BSA in TBS-T and then incubated with secondary HRP-conjugated antibodies for 1 h at room temperature. Primary antibodies were: phospho-MLKL (1/500; ab196436; Abcam, Inc.); cleaved caspase-8 (1/1000, 8592, Cell Signaling Technology); p-cJUN (1/1000, 3270, Cell Signaling); cleaved IL-33 (1/1000; AF3626; R&D Systems), heme oxygenase-1 (HO-1, 1/2000, ADI-OSA-150-D, Enzo) and p65 (1/1000, 8242, Cell Signaling Technology). Total protein content for loading controls was assessed with Ponceau Red or by means of the fluorescence incorporated into tryptophan amino acids of proteins samples ran in Stain-Free^TM gels (BioRad).