C0745

Static activity measurements of Cdk5/p25 phosphorylation of histone H1 were performed by suspending 100 ng of the enzyme (C0745; Sigma Aldrich, MO) in 50 μL of 1× kinase buffer to obtain a final concentration of 18.5 nM. The Cdk5/p25 concentration was reduced five-fold for dynamic measurements to obtain a final concentration of 3.7 nM. A stock solution of 5× kinase buffer was 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) and diluting further to form 1× kinase buffer. Solutions of the substrate protein were prepared by adding 2 mg/mL of histone H1 (10223549001; Sigma Aldrich, MO) to the assay to obtain the final concentrations as described in the main text. The phosphorylation reaction was triggered with a cocktail of DTT and ATP. The final concentration of the ATP and DTT solution was adjusted to 250 μM and 5 mM in DIW respectively.

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.