FIG. 3.

Transient-transfection assays using the 5′ noncoding region of the human ChAT gene driving the luciferase reporter gene. A schematic showing the 5′ noncoding region of the human cholinergic gene and the constructs generated from this region is shown on the bottom. The position of the VAChT gene is indicated, while R represents the first exon of the cholinergic gene. The positions of restriction sites for EcoRI, NheI, and XhoI are shown. Constructs were named on the basis of the restriction fragment inserted into the pXP2 vector, with N corresponding to the NheI site, X corresponding to the XhoI site, and E corresponding to the EcoRI site. PXP2EX-m contains a mutated inactive NRSE. H89 is a PKA inhibitor. Constructs were transiently transfected into the indicated cell lines as described in Materials and Methods. After 48 h, cells were harvested, and extracts prepared from them were subsequently assayed for luciferase activity. Luciferase activity was normalized to β-galactosidase activity.