Sc 81178

Cells were lysed with NP-40 lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP-40) containing 1 mM PMSF (Sigma) and centrifuged to remove debris. Protein concentration in supernatant was assayed using a Quick Start Bradford Protein Assay reagent (Bio-Rad). Each sample containing 90 μg of protein was resolved on 3–20% SDS-PAGE. Upon transfer, Western blot was done with mouse monoclonal anti-utrophin (MANCHO03 clone 8A4, developed by Glenn E. Morris and obtained from the Developmental Studies Hybridoma Bank at the University of Iowa), rabbit anti-eIF4G (sc-11373, Santa Cruz Biotechnology) and mouse anti-β-actin (sc-81178, Santa Cruz Biotechnology) antibodies.

Livers were homogenized using a Polytron homogenizer in a Tris-NP40 buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40) supplemented with a protease inhibitor cocktail (Roche Diagnostics). The homogenates were incubated on ice for 10 min and centrifuged at 10000 g for 10 min to remove tissue debris. Fifty µg of proteins were run on SDS-PAGE mini-gels at the appropriate concentration of acrylamide and transferred onto a polyvinylidene difluoride membrane. Membranes were blocked (1 h at room temperature) with a 5% skim milk in 1×TBST (Tris-buffered saline Tween-20: 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.2% Tween-20) solution and probed with an antibody raised against CYP2E1 (1:1000 dilution, rabbit polyclonal ab84598, (R, H), Abcam, Cambridge, UK) or ß-actin (1:200 dilution, rabbit polyclonal sc-81178 (H, M, R, Hm) Santa Cruz Biotechnology INC, Dallas, TX, USA) overnight at 4 °C. After washing with TBST, blots were incubated at room temperature (1 h) with the appropriate secondary antibody coupled to horseradish peroxidase and washed again. Antibody-bound protein was revealed using the ECL reagent (Thermo Scientific). Films were scanned and analyzed using Image J software. All blots were corrected for loading using ß- actin expression.

Protein levels of soleus muscle of CC and CT samples were analyzed by Western blotting using antibodies specific for collagen I (1:100) (sc-25974, Santa Cruz) and collagen III (1:5000) (ab6310, Abcam). Protein levels were normalized by the endogenous β-actin (1:1000) (sc-81178, Santa Cruz). Muscle protein was extracted using Tris-Triton buffer (10 mM Tris pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate) containing Protease Inhibitor Cocktail (Sigma- Aldrich, USA) and quantified by the Bradford method^{72 (link)}. Samples were separated on a polyacrylamide gel and then transferred to a nitrocellulose membrane. After blockage, membranes were incubated with the primary antibody. Membrane was washed with TBS-T and incubated with secondary peroxidase-conjugated antibody (1:2500). Super Signal® West Pico Chemiluminescent Substrate (Pierce Protein Research Products, Rockford, USA) was used to detect bound antibodies.