Fig. 6. Phosphorylation of S119 by PKA generates a 14-3-3 binding motif. (A) Wild-type and S119A mutated recombinant PCTAIRE-1 proteins were bound to 9E10 beads, incubated either in the presence or absence of 1U PKA, followed by incubation with GST-tagged 14-3-3 or isoforms. The beads were washed, and the bound material was analysed by western blotting using antisera against PCTAIRE-1 (top panel) and GST (bottom panel). In lanes 5 and 6, 10% of the input of the respective 14-3-3 proteins were applied. (B) Constructs encoding wild-type or mutated YFP-tagged PCTAIRE-1 were transfected into Neuro-2A cells. Two days after the transfection, the cells were lysed, the YFP-tagged PCTAIRE-1 proteins were immunoprecipitated using a rabbit serum against GFP, and the bound material was checked for the presence of 14-3-3 proteins and PCTAIRE-1 by western blotting using antisera against 14-3-3-proteins (pan) and PCTAIRE-1, respectively.

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