S6508

Mouse intestinal mucosal cells were collected by scraping from mouse colon, including proximal and distal colon, as previously described.^{28 (link)} Briefly, mouse mucosal cells were lysed in lysis buffer (1% Triton X-100 (Sigma-Aldrich, X100), 150 mM NaCl (J.T. Baker 3624–19), 10 mM Tris (Fisher Scientific, BP152-5) pH 7.4, 1 mM EDTA (Fisher Scientific, BP120-1), 1 mM EGTA (Sigma-Aldrich, E3889) pH 8.0, 0.2 mM sodium ortho-vanadate (Sigma-Aldrich, S6508) and protease inhibitor cocktail (Roche Diagnostics, 118,367,001). Cultured cells were rinsed twice in ice-cold Hanks’ balanced salt solution (Sigma-Aldrich, H1387), lysed in protein loading buffer (50 mM Tris, pH 6.8, 100 mM dithiothreitol (Amresco, 0281), 2% SDS (Sigma-Aldrich, L3771), 0.1% bromophenol blue (IBI Scientific, IB74040) and 10% glycerol (Sigma-Aldrich, G5516)) and sonicated (Branson Sonifier, 250). Equal amount of protein was separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose (Bio-rad, 162–0112) and immunoblotted with primary antibodies: anti-GFAP (Abcam, ab53554), SMMHC (Abcam, ab53219), human SOD-1⁵⁸ or β-actin (Sigma-Aldrich, A1978) antibodies and visualized by ECL chemiluminescence (Thermo Scientific, 32,106). Membranes probed with more than one antibody were stripped before re-probing. Western blot bands were quantified using Image Lab 4.01 (Bio-Rad).