Tissue Lysate Enzymatic Assays

For enzymatic assays, lysates of colonic and hepatic tissues were prepared in Tris buffer (100 mM Tris, 300 mM KCl, 0.1 % Triton X-100, pH 7.0, Calbiochem® protease inhibitor cocktail II (Merck Millipore, Darmstadt, Germany)) using a TissueLyzer (2 × 30 sec; 30 Hz; Qiagen, Hilden, Germany), centrifuged (14.000 x g, 30 min, 4°C) and stored at -80°C until further analysis. Total GPx and total TrxR activities were determined as previously described [47 (link)]. GPx4 activities were measured according to the measurement of total GPx activity, but by applying the specific substrate phosphatidylcholine hydroperoxide (PCOOH, 1.25 mM) instead of 0.00375 % H₂O₂ [24 (link)].