Cdk5/p25 Kinase Activity Assay

The activity of Cdk5/p25 was estimated by measuring the phosphorylation of histone H1 as reported previously.^{8 (link)} All measurements were performed with 18.5 nM of Cdk5/p25 (C0745; Sigma Aldrich, MO) in 1× kinase buffer to match physiological conditions using a volume of 50 μL. The substrate protein histone H1 (10223549001; Sigma Aldrich, MO) was suspended in deionized water at a stock concentration of 2 mg/mL and further diluted as described in the in the Results section. Substrate phosphorylation was initiated with a mixture of dithiothreitol (DTT) and adenosine triphosphate (ATP) with final concentrations of 250 μM and 5 mM respectively. The measurements were buffered using 5× kinase buffer, prepared by suspending 25 mM β-glycerol (G9422; Sigma Aldrich, MO), 50 mM MgCl₂ (5980; Millipore, MA), 5 mM EGTA (E0396; Sigma Aldrich, MO), 2.4 mM EDTA (1002264786; Sigma Aldrich, MO), 1.25 mM MOPS (M1254; Sigma Aldrich, MO) in deionized water (DIW). The kinase buffer was then diluted in DIW to form the 1× kinase buffer used in the assays.