Camp dependent protein kinase pka

Recombinant P301S Tau was phosphorylated as described previously (50 (link)). Two μm Tau was incubated for 6 h at 30 °C with 1 unit/ml cAMP-dependent protein kinase (PKA) (New England Biolabs) in 25 mm Tris-HCl, pH 7.4, 0.1 mm EGTA, 2 mm 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 10 mm magnesium acetate, 2 mm ATP, and 50 μg/ml heparin (Sigma). After 6 h, 1 unit/ml stress-activated protein kinase 4 (SAPK4) (Millipore) was added, and the reaction was allowed to proceed for an additional 6 h. The samples were then incubated for 5 min at 95 °C, before centrifugation for 15 min at 100,000 × g. The supernatants were used.

cAMP-dependent protein kinase (PKA) and lambda protein phosphatase (λPP) were purchased from New England Biolabs.