| Note |
The homogenate supernatant, membrane, and cytosolic fractions of liver were prepared as follows. The livers of control and 100 mg/kg/day groups were homogenized individually in 3 fold volume of ice cold 50 mM Tris-HCl buffer (pH7.4) with 154 mM potassium chloride and PhosSTOP (Roche Diagnostics). The homogenates were centrifuged at 10,000 g, and the homogenate supernatant fractions were obtained from the supernatants. A part of the homogenate supernatant fractions were further ultra-centrifuged at 105,000 g to obtain cytosolic fractions from the supernatants. The membrane fractions were prepared from the pellet after washing in 100 mM potassium phosphate buffer (pH7.4) with PhosSTOP. Each fraction was diluted with the buffer at 1 µg/mL after protein determination by DC protein assay (Bio-Rad Laboratories, Inc.) with albumin as a standard. |