“…The results in Fig 2L represented 8 repeats for EEC culture experiment. After homogenization procedure and preliminary centrifugation (17K x g at 4°C for 15 minutes) to remove mitochondria, cell membranes, nucleus and intact cells, the supernatant from EECs were sedimented at 145K x g for 2 hours (CP80WX, Hitachi Koki Co., Ltd., Hitachi, Tokyo, Japan) to collect microsomes, then were sonicated and stored in -20°C for Western blotting following the previously described procedure [ 19 ]. In brief, microsomes (10 μg protein/sample as determined by the method of Bradford reagent (B6916, Sigma-Aldrich, according to the manufacturer’s protocol)) were subjected to 10% SDS-PAGE gel, and the separated proteins were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (NEF1002001PK, PerkinElmer, Waltham, MA, USA).…”