Gpx1 2

Cells were suspended in lysis buffer (20 mM Tris-HCl, pH 7.8, 50 mM NaCl, 5 mM EGTA, and 1% v/v Triton X-100) containing freshly added protease and phosphatase inhibitors (Roche). Lysates were clarified by centrifugation at 4°C, and protein concentration was determined by Bio-Rad protein assay.
To verify the protein contents of the BMSC-ECM after alkaline detergent extraction, BMSC-ECM were scratched from culture plate and dissolved in buffer containing 100 mM Tris-HCl (pH 6.8), 200 mM dithiothreitol and 4% SDS. Samples were homogenized by pass through 27 g needle for at least 7 times, incubated on ice for 60 minutes, cleared by centrifugation and subjected to protein concentration assessment. Then 0.2% glycerol and 0.2% bromophenol blue (final concentration) were added and samples were heated at 95°C for 5 minutes.
The following antibodies are used: phosphor-AKT Ser 473 and were from Cell Signalling. γ-H2AX, osteopontin, β-actin, MnSOD2, BCL-2, and BCL-xL were from Millipore. p27, p21, collagen 1, fibronectin, MCL-1, Gpx1/2, were from Santa Cruz Biotechnology.

Protein samples were prepared from liver homogenates in Laemmli sample buffer, run on SDS-polyacrylamide gels (4-15% TGX stain-free gel, Bio-Rad), and transferred to the polyvinylidene difluoride (PVDF) membrane. The membranes were blocked, incubated with primary antibodies overnight at 4 °C, followed by secondary antibodies, and developed using the chemiluminescence imaging system (Bio-Rad). Following primary antibodies were used: OPA1 (BD Biosciences, 612606; 1:1000); caspase-3 (Cell Signaling, 9662; 1:1000), PARP-1 (Cell Signaling, 9542; 1:1000), β-actin (Sigma, A1978; 1:40000), TOM20 (Proteintech, 11802-1-AP; 1:1000), cytochrome c (BD Biosciences, 556432; 1:5000), elF2α (Cell Signaling, 9722; 1:1000), phospho-elF2α (Cell Signaling, 9721; 1:1000), FGF21 (Proteintech, 26272-1-AP; 1:1000), LC3 A/B (Cell Signaling, 4108; 1:1000), PGC1α (Invitrogen, PA5-38022; 1:500), OMA1 (Santa Cruz, sc-515788; 1:100), and mitochondria total OXPHOS rodent WB cocktail (Abcam, ab110413; 1:1000). JNK (Cell Signaling, 9252; 1:1000), p-JNK-Thr183/Tyr185 (Cell Signaling, 9255; 1:500), MCU (Sigma, HPA016480; 1:1000), NCLX (Proteintech, 21430-1-AP; 1:1000), CypD (Proteintech, 18466-1-AP; 1:1000), MnSOD (BD Biosciences, 611580; 1:1000), GPx1/2 (Santa Cruz Biotechnology, sc-133160; 1:200), and CYP2E1 (Proteintech, 19937-1-AP; 1:300).

Lieber-DeCarli regular control liquid diets (D710027) and Lieber-DeCarli regular ethanol liquid diets (D710260) were purchased from Dyets Inc. (Bethlehem, PA, USA). Maltose dextrin was purchased from BioServ (Flemington, NJ, USA). Nonidet^TM P 40 substitute and 200-proof ethyl alcohol were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Antibody against Cytochrome P450 2E1 (CYP2E1) was purchased from Invitrogen (Waltham, MA, USA). Antibodies against superoxide dismutase (SOD), catalase (CAT), AMP-activated kinase (AMPK), and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against aldehyde dehydrogenase 2 (ALDH2), sirtuin 1 (SIRT1), sterol regulatory element-binding protein 1c (SREBP-1c), peroxisome proliferator-activated receptor α (PPARα), and glutathione peroxidase 1/2 (GPx 1/2) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Malondialdehyde (MDA, ab118970), GPx (ab102530), and triglyceride (TG, ab65336) assay kits were purchased from Abcam (Cambridge, UK).