Fig. 9.

Agonist-stimulated effects on pCRE-luciferase reporter gene activity and c-Fos in SH-SY5Y cells. Sub-confluent SH-SY5Y cells were transfected with the pCRE-luciferase reporter construct as described in Materials and methods. Cells were treated with MCh (100 μM), bradykinin (30 μM), PDBu (1 μM) or forskolin (10 μM)/IBMX (500 μM) for 1 h, followed by further 5 h incubation in serum-free medium (A). Time-dependency of increases in CREB-dependent luciferase activity after stimulation of cells with MCh (100 μM) for the times indicated (B). Concentration-dependency of CREB-dependent luciferase activity after 1 h stimulation with MCh at concentrations indicated (C). In addition, cells were stimulated with MCh (100 μM), bradykinin (30 μM) or PDBu (1 μM) for 2 h prior to lysis and c-Fos determination (D; see Section 2). A representative immunoblot showing c-Fos immunoreactivity is shown below the histogram. Data are normalized to the luciferase activity or c-Fos immunoreactivity detected after assay buffer addition alone, and are presented as means ± S.E.M. for 3 independent experiments each performed in duplicate.