Statistical Analysis All graphical data were prepared

Statistical Analysis: All graphical data were prepared and statistical analyses were performed using GraphPad Prism. Statistical significance between the differences of means was determined by performing a One-way Analysis of Variance followed by the Bonferroni\'s post-hoc test.
Results CDK5 is phosphorylated in an activation-dependent manner: Reversible phosphorylation is a key regulatory mechanism to control kinase activity. In this study, we wished to identify specific sites and study phosphoregulation of CDK5. Since phosphorylation is known to retard electrophoretic mobility of modified proteins, we first determined if we could detect phosphomodified CDK5 in cell lysates using a mobility shift based western blot. Cos7 Phos-tag Biotin BTL-105 sale were transfected with CDK5 - either WT or D144N (catalytically inactive) mutant with or without its cognate activator, p35. Cos7 cells do not have detectable levels of endogenous p35, therefore, they serve as an excellent system to control CDK5 activation by simply including or omitting p35 from the transfections [17]. Cells were lysed 48 h post-transfection and lysates were analyzed by western blotting. As shown in Fig. 1A, when expressed without p35 (lanes 1 and 3), both WT and D144N CDK5 were detected as a single band. D144N CDK5 also showed a single band even in the presence of p35 (lane 4). In contrast, WT CDK5 showed slower migrating bands in addition to the main band when co-expressed with p35 (lane 2) suggesting that CDK5 is post-translationally modified in a p35 i.e. activation dependent manner. p35 is also a substrate for CDK5 [17] and it showed a broader/more smeared band (suggestive of its phosphorylation) when co-expressed with WT but not D144N CDK5 further confirming that WT CDK5 was activated in the presence of p35. To ascertain that the slower migrating bands represented phosphorylated CDK5, we performed a phosphatase assay. Lysates of cells co-expressing p35 and either WT or D144N CDK5 were immunoprecipitated using an anti-p35 antibody. The immunoprecipitates were then dephosphorylated using SAP along with appropriate controls. As shown in Fig. 1B (Top), D144N CDK5 samples showed a single band in all three conditions (lanes 1–3) suggesting the absence of any post-translational modification. The WT CDK5 on the other hand, showed the slower migrating bands in the control lane without SAP (lane 4). Upon treatment with SAP, the slower migrating bands disappeared and instead showed a single band (lane 5) comparable in mobility to that of D144N CDK5 suggesting that the mobility shift seen in lane 4 was due to phosphorylation of CDK5. As a negative control, when SAP was added along with EDTA (SAP inhibitor), the slower migrating phosphorylated bands of CDK5 were restored (lane 6). As expected, we also observed the same pattern for p35 i.e. modified p35 bands in lanes 4 and 6 but not in lanes 1–3 and 5. Together, these results confirmed that the bands showing retarded electrophoretic mobility represent phosphorylated CDK5/p35 and that the phosphorylation of CDK5 is dependent on its catalytic activity.