In Vitro CDKL5 Kinase Assay

Methods used for these assays have been described.^{28 (link)} Briefly, human FLAG-tagged CDKL5 WT or kinase dead (KD, CDKL5 K42R) constructs were subcloned into pT7CFE1-CHis plasmid (Thermo Fisher). These constructs were then used for in vitro translation using a HeLa cell lysate-based Kit (1-Step Human Coupled IVT Kit—DNA, 88881, Life Technologies). The in vitro-translated proteins were then purified using His Pur cobalt spin columns (Thermo Scientific). For in vitro kinase assays, recombinant CDKL5 and myelin basic protein (Active Motif, 31314) as a substrate were incubated in a kinase buffer (Cell Signaling, 9802) supplemented with or without 50 µM adenosine 5′-triphosphate (ATP) at 30 °C for 30 minutes followed by kinase assays using ADP-Glo Kinase Assay kit (Promega). Data were analyzed using GraphPad Prism 9. Each assay was run in triplicate (n = 3) and mean values are graphed in Figure 7. All error bars in Figure 7 are standard deviation (SD). Statistical analysis was done using one-way ANOVA with Dunnett’s multiple comparisons test. Statistical methods and p-values are mentioned in the Figure 7 legend. Thresholds for significance were placed at ***p <0.0001. Non-significant statistics were not indicated.