Horseradishperoxidase conjugated anti rabbit or mouse igg antibody

Total cellular protein was extracted by suspending the cells in sodium dodecyl sulphate (SDS) lysis buffer together with freshly added proteinase inhibitor cocktail (Nacalai tesque, Kyoto, Japan). Lysates were incubated on ice for 30 min with intermittent mixing and then centrifuged at 12,000 rpm for 10 min at 4 °C in an Eppendorf centrifuge. The supernatant was recovered and the protein concentration was determined using the Micro BCA Protein Assay Kit (The Thermo Scientific Pierce, Rockford, IL). Western blot was performed using the enhanced chemiluminescence system. Briefly, extracted cellular proteins were separated by 10% SDS polyacrylamide gels and electrotransferred onto polyvinylidine difluoride membranes. After blocking with 3% bovine serum albumin in PBS, the membranes were incubated with primary antibody for 1.5 h at room temperature or at 4 °C overnight. After washing, the membranes were probed with horseradishperoxidase-conjugated anti-rabbit or -mouse IgG antibody (Cell Signaling, Beverly, MA), and the bands were visualized by using the enhanced chemiluminescence system (Nacalai Tesque, Kyoto, Japan). The chemiluminescent signal was captured with a Fujifilm luminescent image LAS-1000 analyzer (Fujifilm, Tokyo, Japan) and quantified with the Image J software (http://rsb.info.nih.gov/ij). To confirm equal loading of proteins, the membranes were probed for β-actin.