Fig. 3. Phosphorylation of S153 by PKA inhibits PCTAIRE-1 kinase activity. (A) Constructs encoding wild-type or mutated YFP-tagged PCTAIRE-1 were transfected into Neuro-2A cells. 48 hours after transfection, the cells were lysed, the YFP-tagged PCTAIRE-1 proteins immunoprecipitated using a rabbit serum against GFP, and the bound material was assayed for kinase activity towards MBP. (top panel) Coomassie-blue-stained gel; (middle panel) autoradiograph at the position of MBP; (bottom panel) radioactivity (cpm) in the MBP bands. (B) Constructs encoding wild-type and YFP-tagged PCTAIRE-1 were transfected into Neuro-2A cells. 48 hours after transfection, the cells were treated with 10 µM forskolin to stimulate PKA for 2 hours, or left untreated. Cells were lysed, the YFP-tagged PCTAIRE-1 proteins were immunoprecipitated using a rabbit serum against GFP and assayed for kinase activity towards MBP. (top panel) Coomassie-blue-stained gel; (middle panel) autoradiograph at the position of MBP; (bottom panel) radioactivity (cpm) in the MBP bands. (C) Constructs encoding wild-type and mutated YFP-tagged PCTAIRE-1 were transfected into Neuro-2A cells. 48 hours after transfection, the cells were lysed, the YFP-tagged PCTAIRE-1 proteins immunoprecipitated using a rabbit serum against GFP, and the bound material was incubated in the presence or absence of PKA, washed, and then assayed for kinase activity towards MBP. (top panel) Coomassie blue stained gel; (middle panel) autoradiograph at the position of MBP; (bottom panel) radioactivity (cpm) in the MBP bands.