Recombinant full length human cdk5 and p35

We identified a putative consensus motif for Cdk5 phosphorylation in P2X2aR protein sequence by using prediction websites Motif-Scan http://scansite.mit.edu/motifscan_seq.phtml and NetPhos2.0 http://www.cbs.dtu.dk/services/NetPhos/ In order to evaluate potential Cdk5 phosphorylation sites, 10 amino acid residues of P2X2aR peptides were tested in an in vitro Cdk5 kinase assay. Recombinant full-length human Cdk5 and p35 (Sigma) were incubated in 50 μl of kinase assay buffer [100 mM Tris-HCl (pH 7.4); 50 mM MgCl₂; 5 mM EDTA; 50 μM NaF; 5 μM Na₂VO₃; 5 mM DTT] containing either 10 μg of histone H1 (Sigma) or P2X2aR peptides (mouse P2X2aR, KVRTPRHPSS; human P2X2aR, KVCTPSHPSG; rat P2X2aR, KVRTPKHPSS; and non-related peptide, IGQSPFHGDD). Kinase assays were carried out at 30°C for 60 min by adding 5 μCi of [γ-³²P] ATP (0.5 mM). The peptide assay was stopped by adding 10% trichloroacetic acid to precipitate proteins. 20-μl aliquots of trichloroacetic acid supernatant were transferred onto P81 phosphocellulose squares (spotted in duplicates), air-dried, and washed five times for 15 min each in 75 mM phosphoric acid and once in 95% ethanol. After air drying, squares were transferred to vials containing Bio-Safe II scintillation fluid (Research Products International, Mount Prospect, IL) for counting in a Beckman Coulter (Fullerton, CA) scintillation counter (model SL 3801).