Microcystin lr

Flag-PKN1 and Flag-PKN2 were transiently transfected into 293T cells, with torin or rapamycin added 4 h before cell harvest at where indicated. Cells were lysed in a buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 1 mM PMSF, 2 mM DTT, 5 mM EDTA, 5 μg/mL each of aprotinin/leupeptin/pepstatin, and 1:100 dilution of phosphatase inhibitor cocktail #1 and #2 (Roche). The cell extracts were incubated with M2-agarose (Sigma A2220) at 4°C for 4 h, and the beads were washed five times with the wash buffer carrying 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1 mM DTT, 0.1 mM EDTA, 1 μg/mL each of aprotinin/leupeptin/pepstatin, 2 mM sodium vanadate, and 0.4 μM Microcystin-LR (Enzo Life Sciences, Farmingdale, NY, Inc. #350-012-C100). The beads were either used directly in the kinase assays, or further eluted at room temperature with 20 μg/mL of Flag peptide in the wash buffer. The kinase assays were typically carried out at 30°C for 15–30 min in a 25 μL volume reaction of 50 mM Hepes, pH 7.0, 2 mM MgCl², 1 mg/mL BSA, 1 mM EGTA, 1 mM DTT, 0.1 mM ATP, 20 mM β-glycerophosphate, 0.15 mM sodium vanadate, 5 μg/mL each of aprotinin/leupeptin/pepstatin, 0.004% SDS, 2–5 μg substrate, the kinase, and 0.1 μL of γ-³²P-ATP. The reactions were stopped by the addition of SDS-PAGE loading buffer (1× final concentration).