PKC kinase assay

COS-7 cells were transfected with the indicated expression vectors or the control vector pcDNA3, as described earlier, and cellular proteins were extracted by cell lysis in PKC extraction buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% Tween-20, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol) that contained protease inhibitors (10 mg/ml aprotinin, 10 mg/ml leupeptin, 0.1 mM PMSF) and phosphatase inhibitors (1 mM NaF, 0.1 mM Na3VO4, 10 mM b-glycerophosphate). HA-tagged PKC proteins were immunoprecipitated from 300 mg of cell extracts using 3 mg of the anti-HA antibody and 30 ml of protein G-sepharose, after a 3 hour incubation at 4oC. The immunoprecipitates were washed twice with PKC extraction buffer and then twice with IP kinase buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM DTT, 2.5 mM EGTA, 1 mM NaF, 0.1 mM Na3VO4, 10 mM b-glycerophosphate) and resuspended in 20 ml of IP kinase buffer. The kinase assay was initiated by adding 40 ml of IP kinase buffer containing 10 mg of GST-MARCKS substrate and 5 mCi of [g-32P]ATP. The reactions were performed at 30oC for 30 min. The reactions were terminated by adding SDS sample buffer and boiled for 5 min. The reaction products were analyzed by SDS-PAGE and autoradiography. Recombinant GST-MARCKS proteins were expressed in E.coli strain BL21(DE3)/LysS and purified to homogeneity using glutathione S-sepharose beads (Pharmacia).