Cdk5 p25

Phosphorylation of 725 nM r-synGAP-α1 by 10 nM CaMKII was carried out as previously described (Walkup et al., 2015 (link)) in reaction mixtures containing 50 mM Tris-HCl, pH 8.0, 10 mM MgCl₂, 0 or 0.7 mM CaCl₂, 0.4 mM EGTA, 500 μM [γ-³²P]-ATP (375 cpm/pmol) (6000 Ci/mmol, Perkin Elmer, Waltham, MA; catalog no. BLU002Z/NEG002Z), 0 or 3.4 μM calmodulin, 10 mM DTT. Phosphorylation of 286 nM r-synGAP-α1 and 4.3 μM Histone H1 (New England Biolabs, Ipswich, MA; catalog no. M2501S), by CDK5/p35 (EMD Millipore, catalog no. 14-477M) or CDK5/p25 (EMD Millipore, catalog no. 14–516) was carried out in the same reaction mixture containing 110 nM CDK5/p35 or CDK5/p25 but no CaMKII. After fractionation by SDS-PAGE, phosphorylated proteins were quantified with a Typhoon LA 9000 phosphorimager (GE Healthcare Life Sciences, Pittsburgh, PA) as previously described (Walkup et al., 2015 (link)). Relative densities were converted to pmol phosphate by comparison to densities of standard amounts of [γ-³²P]-ATP. The stoichiometry of phosphorylation was calculated by dividing mol of incorporated phosphate by mol of r-synGAP-α1 loaded per lane.

FLAG-tagged wild-type or mutant Drosha was transfected into HEK-293T cells as described previously^{25 (link)}. The recombinant active Cdk5/p25 protein was purchased from Millipore Sigma (14-477). The Cdk5 kinase assay was performed as previously described with modifications^{27 (link)}. Briefly, for the in vitro kinase assay, 6.4 μl of 5× kinase reaction buffer, 7.2 μl of ddH₂O, 0.16 μl of phosphatase inhibitors, 2 μl of 10 mM ATP (A2383, Sigma), and 10 μl of 0.1 μg/μl purified FLAG-tagged proteins were incubated with 5 μl of 0.1 μg/μl Cdk5/p25 at 30 °C for 30 min. To stop the reaction, 5× loading buffer was added to the tube, and the sample was boiled at 100 °C for 5 min.