CDK5 Phosphorylation and Deacetylation Assays

In vitro phosphorylation assays were performed as previously described^{60 (link)}. Briefly, purified recombinant His-CDK5 WT or His-Ac-CDK5 (1 µg) was incubated at 30 °C for 30 min with 1 µg of histone H1 protein (Merck, #382150) and 1 µg of p25-His in 1 × kinase buffer (40 mM Tris-HCl pH 7.6, 2 mM DTT, 5 mM MgCl₂, 1 mM NaF, 50 µM unlabeled ATP) in the presence of 5 µCi [γ-³²P]ATP or cold ATP. Phospho-H1 signals were visualized by autoradiography or immunoblotting with a rabbit polyclonal anti-phospho-histone H1 antibody (1:1,000; Millipore, #06-597). Transiently transfected HEK293 cells and hippocampal neurons were lysed, immunoprecipitated with anti-CDK5 antibody and processed for in vitro phosphorylation assays. To assess ATP binding affinity, purified recombinant His-CDK5 WT and His-Ac-CDK5 (1 μg) were reacted for 2 hrs with 20 μl of ATP-conjugated agarose (Jena Bioscience, #AC-101) as previously described^{61 (link)}. For the in vitro deacetylation assay, immunoprecipitated FLAG-tagged SIRT1 or SIRT2 was incubated with 0.5 µg of purified recombinant Ac-CDK5 supplemented with 5 mM β-nicotinamide adenine dinucleotide (NAD⁺, Sigma, #N7004) at 30 °C for 2 hrs as previously described^{62 (link)}.