Fig. 1.

Time-courses and concentration-dependencies for receptor-mediated phosphorylation of the transcription factor CREB in SH-SY5Y cells. Time-dependent increases in phospho-CREB immunoreactivity after stimulation of cells with either methacholine (1 μM; A) or bradykinin (300 nM; B). A representative immunoblot for phospho-CREB and total CREB is shown beneath each graph (Ctr, buffer addition; 1′–30′, times (in min) of exposure to agonist; Fk, response to addition of 10 μM forskolin/500 μM IBMX for 10 min). MCh activation resulted in significant (^*p < 0.05; ^**p < 0.01; ^***p < 0.001) increases in phospho-CREB between 1 and 30 min stimulation, while activation by bradykinin resulted in a significant increase in phospho-CREB only after 1 min of stimulation. Concentration-dependent increases in phospho-CREB immunoreactivity were assessed at a 5 min time-point for M₃ mACh receptor (C) or B₂ bradykinin receptor (D) stimulation with the indicated concentrations of agonist. For each individual experiment, data have been normalized to the increase in phospho-CREB observed in the presence of a maximal concentration of agonist (100 μM methacholine and 30 μM bradykinin, respectively). For each panel, data are shown as means ± S.E.M. for at least 3 independent experiments.