Kinase activity assay

Cdk5 was immunoprecipitated using anti-Cdk5 antibody (sc-173, Santa Cruz Biotechnology) and Protein G Agarose beads (Sigma-Aldrich). Beads were resuspended in 50 μl kinase buffer (50 mM HEPES pH 7.0, 10 mM MgCl2, 1 mM DTT, 1 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 3 mM β-glycerophosphate, 4 mM Complete EDTAfree). An aliquot of 2 μM ATP, 10 μCi 32P-γ-ATP (Hartmann Analytic, Braunschweig, Germany) and 0.05 μg μl⁻¹ histone H1 (Type III from calf thymus, Sigma-Aldrich) were added and samples were incubated at 30 °C for 20 min before termination by boiling with 5 × SDS sample buffer (5 min, 95 °C). Electrophoresis and autoradiography was performed.