Kinase reaction buffer

HEK-293 cells expressing recombinant FLAG-CPEB3a protein were harvested 36 h post-transfection by scraping in ice-cold lysis buffer containing 10 mM HEPES pH 7.4, 200 mM NaCl, 2 mM EDTA, 1% Tx-100, Complete Mini protease inhibitors (1 tablet/10 ml) and Phos-Stop phosphatase inhibitors (1 tablet/10 ml). Lysates were sonicated for 10 min in ice-cold sonication bath and spun down (15 min/17.000 rcf). Supernatants were mixed with 50 μl of FLAG M2 agarose beads (Sigma), previously blocked with 3% BSA, and incubated for 2 h at 4°C on a rotator, followed by 5x washing with lysis buffer. CaMKII was preactivated at 30°C for 30 min in kinase reaction buffer (New England Biolabs) supplemented with 1.2 μM calmodulin and 2 mM CaCl₂. Beads were mixed with kinase buffer containing 1 mM ATP and PKA-C_α (5000 U, New England Biolabs), pre-activated CaMKII (1000 U, New England Biolabs), or the corresponding buffer without enzyme. Sample was incubated for 30 min at 30°C with rotation and then mixed with 50 μl of NuPage reducing sample buffer and separated by SDS PAGE.

Five μg of rCP or kinase specific substrates and 0.5 μg of kinase [purified NbCKII or commercial mammal kinases PKA or CaMKII (New England Biolabs)], were incubated in 10 μl reactions containing 1X kinase reaction buffer (New England Biolabs), 3 μCi γ-³²P-ATP (Perkin Elmer) and 200 μM unlabeled ATP. To inhibit PKA phosphorylation, 1 μM of PKA specific inhibitor H-89 [5-Isoquinolinesulfonamide, N-(2-((3-(4-bromophenyl)-2-propenyl)amino)ethyl)] was added. After incubation at 30 °C for 30 min, the reactions were quenched by addition of 5X SDS sample buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 50% Glycerol, 0.5% Bromophenol blue, 5% β-mercaptoethanol) and boiled for 5 min. Then samples were separated by 12.5% SDS-PAGE followed by autoradiography.