Rediplate 96 enzchekr tyrosine phosphatase assay kit

After treatment with SC-43, cell protein extracts were incubated with anti–SHP-1 antibody in immunoprecipitation buffer [20 mM Tris-Hcl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 1% sodium deoxycholate] overnight. Protein G-Sepharose 4 Fast Flow (GE Healthcare Bio-Science, NJ) was added to each sample, followed by incubation for 3 hours at 4°C with rotation. A Rediplate 96 Enzchekr Tyrosine Phosphatase Assay Kit (R-22067) was used for SHP-1 activity assay (Molecular Probes, Invitrogen, CA). For the examination of the SHP-1 activity in the SHP-1–containing immunoprecipitation of cellular extract, 1 mg of whole-cell lysates was incubated with anti–SHP-1 antibody in immunoprecipitation buffer overnight. Then, samples were incubated with Protein G-Sepharose 4 Fast Flow for 3 hours at 4°C with rotation and finally treated with SC-43 for 2 hours at 4°C. The SHP-1 activity in SHP-1–containing immunoprecipitation of cellular extract following SC-43 treatment was determined by using a Rediplate 96 Enzchekr Tyrosine Phosphatase Assay Kit.