Kinase assay

For a typical reaction, 5 μl of 3 × kinase buffer (90 mM HEPES, 150 mM potassium acetate, 15 mM MgCl₂) was mixed with 150 ng of GST-MARK4 (Sigma) and 150 ng of lab-made recombinant 4R2N hTau followed by addition of AC, MB or OS at the indicated concentrations. ATP was added last to a final concentration of 200 μM. The total volume was brought up to 15 μl with protease free H₂O. The reaction was incubated at 30 °C for 30 min and then terminated by adding 4 × LDS buffer with 2-mercaptoethanol. After incubation at 70 °C for 10 min, the samples were analyzed by Western blot.