Western lightning plus ecl chemiluminescence kit

Cells were lysed in a 40 μl of lysis buffer containing 1% Triton X-100, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml of aprotinin, and 10 μg/ml of leupeptin. Following incubation (at 4°C for 15 min) and centrifugation at 17,400 x g (at 4°C) for 15 min, supernatants were mixed with equal volume of 2X Laemmli sample buffer and heated at 100°C for 5 min. Separation of membranous and cytosolic fractions from samples was performed using Trident Membrane Protein Extraction kit (Genetex) according to the manufacturer's protocols. Ten microliters of the samples was electrophoresed in 12.5% PAGE gel (Atto) and transferred onto Immnobilon-P membranes (Merck Millipore). The membranes were blocked with 5% skim milk at room temperature for 1 h. Primary antibodies were diluted with Tris-base buffered saline with Tween (TBS-T) and incubated at 4°C for overnight. Subsequently, the membranes were incubated with diluted secondary antibodies at room temperature for 1 h. The transferred proteins detected by antibodies were visualized by the Western Lightning Plus-ECL chemiluminescence kit (PerkinElmer, Inc.) according to the manufacturer's protocols. All the data shown are representatives of results of at least three independent experiments.