After three times washing with lysis buffer and with kinase buffer the immunoprecipitates were subjected to an in vitro kinase reaction with 40 μl of reaction mixture containing kinase reaction buffer supplemented with 200 μ M ATP and 1 μg GSK-3-fusion protein.
Briefly, after washing with cell lysis buffer and kinase buffer, the immunoprecipitates were incubated with a GSK-3 fusion protein substrate and ATP for 30 min at 30°C.
Proteins were extracted from the cell lysates using MAP kinase lysis buffer and the western blot analysis was carried out as previously described (Winn and Heasley, 2004).
For Buffer System I (BSA), the blocking buffer was 1% BSA in PBS, and the kinase reaction buffer contained 1% BSA, 1% NP-40, 100 mM MOPS pH 7.2, 100 mM NaCl, 5 mM MgCl2, 5 mM MnCl2, 1 mM DTT.
After washing, the proteins bound to beads, were resuspended with 20 µl kinase buffer and kinased with recombinant JNK1.
The precipitates were then washed twice with the lysis buffer and twice with kinase assay buffer (25 mM Tris-HCl pH 7.5, 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, and 10 mM MgCl2), and resuspended in 50 µl of kinase assay buffer as the kinase source for the following kinase assay.
Immunocomplexes were washed two times with kinase buffer and kinase reactions were carried-out as described above.
Beads from immunoprecipitations were washed three times in lysis buffer and once in kinase buffer, and kinase assays were performed with recombinant GST-S6 as substrate (1 μg per assay).
Beads were then resuspended in 50 µl kinase buffer, and kinase reaction was carried out for 30 min after addition of 1 µg of substrate and ATP to a final concentration of 200 µM.
20×106 thymocytes were lysed in lysis buffer, and then the creatine kinase activity was determined by the rate of formation of NADPH from creatine phosphate using standardised methods [39].
