Phosstop phosphotase inhibitor cocktail tablet

Cells and tissues were washed with cold PBS on ice and treated with RIPA lysis buffer (Millipore, Temecular, CA) containing a cOmplete protease inhibitor cocktail tablet (Roche, Madison, MI) and a PhosSTOP phosphotase inhibitor cocktail tablet (Roche, Madison, MI). The protein amount was determined using the Bio-Rad protein assay (Bio-Rad). After an equal amount of protein was loaded in each lane, they were separated by 10% (w/v) SDS-PAGE and then transferred to an Immobilon-P PVDF membrane (Millipore). Target proteins were immunodetected using specific primary antibodies and the horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, CA) was used as a secondary antibody. Positive bands were detected using the Supersignal Westpico Chemiluminescent Substrate (Thermo Scientific, Pittsburgh, PA). Antibodies for p-TAK1, TAK, p-IKK α/β, IkB-α, p-JNK, JNK and AKT were obtained from Cell Signaling Technology (Boston, MA), antibodies for p-AKT and HSP90α/β were from Santa Cruz Biotechnology (Santa Cruz, CA) and the antibody for GPR120 was from Novus Biologicals (Littleton, CO).