Tissue homogenate preparation

Tissue pieces (3 mm by 3 mm; liver and pancreas) were homogenized in 200 μl of Cell Extraction Buffer (100 mM tris, 2 mM Na₃VO_4, 100 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, 1 mM EGTA, 0.1% SDS, 1 mM NaF, 0.5% deoxycholate, and 20 mM Na₄P₂O₇), pH 7.4, containing PhosSTOP Phosphatase Inhibitor Cocktail (Roche) and EDTA-free Complete protease inhibitor cocktail (Roche). The homogenates were prepared using a bead beater, and the homogenates were centrifuged at 9000g for 5 min at 4°C. The previous steps were repeated until a clear tissue homogenate was produced. The tissue homogenate protein concentration was quantified using the bicinchoninic acid (BCA) assay using BSA as a standard.