Fig. 5. Does PCTAIRE-1 require a brain-specific partner protein? (A) Gel filtration analysis of an extract from mouse brain. The lysate prepared from one mouse brain was applied on an AcA34 column, PCTAIRE-1 was immunoprecipitated from the individual fractions using mAb G6, and assayed for kinase activity. The reaction mix was then applied on an SDS-PAGE, blotted, and checked with a polyclonal PCTAIRE-1 antibody for the presence of PCTAIRE-1. Subsequently, the blot was exposed for autoradiography. (B) Constructs encoding wild-type, kinase-dead, or S153A mutated YFP-tagged PCTAIRE-1 were transfected into Neuro-2A, HEK 293, CHO, COS-7 and HeLa cells. Two days after the transfection, the cells were lysed, and the YFP-tagged PCTAIRE-1 proteins were immunoprecipitated using a rabbit serum against GFP, and assayed for kinase activity on MBP. (top panel) Coomassie-blue-stained gel; (middle panel) autoradiograph at the position of MBP; (bottom panel) radioactivity (cpm) in the MBP bands. The S153A mutant PCTAIRE-1 protein kinase is active in a wide range of cell lines. (C) Constructs encoding wild-type, kinase-dead, S153A, S119A/S153A mutated YFP-tagged PCTAIRE-1 and YFP-tagged CDK5, were transfected into Neuro-2A and HEK 293 cells. Two days after the transfection, the cells were lysed, and the YFP-tagged proteins were immunoprecipitated using a rabbit serum against GFP, and assayed for kinase activity towards MBP. p21^nck was added to reactions with CDK5 as indicated. (top panel) Coomassie-blue-stained gel; (middle panel) autoradiograph at the position of MBP; (bottom panel) radioactivity (cpm) in the MBP bands. Note that PCTAIRE-1 and CDK5/p21^nck display comparable kinase activity.