Map kinase activation sampler kit

Jurkat T-cells were treated with various stimulations by 2’,4’-dihydroxy-6-methoxy-3,5-dimethylchalcone and disrupted by sonication in a homogenizing buffer (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 2 mM EDTA, 10 mM β-glycerophosphate, and 1 mM Na₃VO₄) containing a complete protease inhibitor (Roche, IN, USA). The homogenates were centrifuged at 10,000 g for 10 min. The supernatants were obtained, and protein concentrations were calculated using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Inc., IL, USA). The protein (20 μg) was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a Hybond-P polyvinylidene difluoride membrane (GE Healthcare). Membranes were blocked in Tris-buffered saline (TBS) containing 50% Block Ace (Dainippon Sumitomo Pharma, Osaka, Japan) and incubated with primary antibodies in TBS containing 10% Block Ace for 1 h at room temperature. MAPK was analyzed. Total or phosphorylated Erk-1/2, p38, and JNK primary antibodies were supplied as a part of a MAP Kinase Activation Sampler Kit (BD Bioscience) and used according to the manufacturer's instructions. The membrane was then incubated with horseradish peroxidase-conjugated secondary antibodies, and the signals were visualized by ECL Plus Western Blotting Detection System (GE Healthcare).