Fig. 4. PCTAIRE-1 kinase activity — substrates and structural requirements. (A) Neuro-2A cells were transfected with S153A mutated YFP-tagged PCTAIRE-1. 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 various substrates. (top panel) Coomassie-blue-stained gel; (middle panel) autoradiograph at the position of MBP; (bottom panel) radioactivity (cpm) in the substrate bands. (B) Neuro-2A cells were transfected with constructs encoding wild-type, mutated and truncated YFP-tagged PCTAIRE-1. Two days after the transfection, the cells were lysed and the YFP-tagged PCTAIRE-1 proteins were immunoprecipitated using a rabbit serum against GFP, washed, and assayed for kinase activity towards MBP. (top panel) Coomassie-blue-stained gel; (second panel) autoradiograph at the position of PCTAIRE-1; (third panel) autoradiograph at the position of MBP; (bottom panel) radioactivity (cpm) in the MBP bands. (C) Neuro-2A cells were transfected with constructs encoding N-terminally truncated, kinase-dead and S153A mutated YFP-tagged PCTAIRE-1. Two days after the transfection, the cells were lysed, and the YFP-tagged PCTAIRE-1 proteins were immunoprecipitated using a rabbit serum against GFP, washed, 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.