| Kinase Assay | Recombinant ROCK-I, ROCK-II, PKN, or citron kinase is expressed in HeLa cells as Myc-tagged proteins by transfection using Lipofectamine, and is precipitated from the cell lysates by the use of 9E10 monoclonal anti-Myc antibody coupled to G protein-Sepharose. Recovered immunocomplexes are incubated with various concentrations of [32P]ATP and 10 mg of histone type 2 as substrates in the absence or presence of various concentrations of either Y-27632 or Y-30141 at 30°C for 30 min in a total volume of 30 μL of the kinase buffer containing 50 mM HEPES-NaOH, pH 7.4, 10 mM MgCl2, 5 mM MnCl2, 0.02% Briji 35, and 2 mM dithiothreitol. PKCa is incubated with 5 μM [32P]ATP and 200 μg/mL histone type 2 as substrates in the absence or presence of various concentrations of either Y-27632 or Y-30141 at 30°C for 10 min in a kinase buffer containing 50 mM Tris-HCl, pH 7.5, 0.5 mM CaCl2, 5 mM magnesium acetate, 25 μg/mL phosphatidyl serine, 50 ng/mL 12-O-tetradecanoylphorbol-13-acetate and 0.001% leupeptin in a total volume of 30 μL. Incubation is terminated by the addition of 10 μL of 43 Laemmli sample buffer. After boiling for 5 min, the mixture is subjected to SDS-polyacrylamide gel electrophoresis on a 16% gel. The gel is stained with Coomassie Brilliant Blue, and then dried. The bands corresponding to histone type 2 are excised, and the radioactivity is measured[1]. |
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