FIGURE 2.

RCAN1.4 transcription is induced by PACAP. A and B, PC12 cells were treated with PACAP (10 nm) for the indicated times, and RCAN1.4 (A) and RCAN1.1 (B) mRNA levels were measured by quantitative real-time PCR. *, p < 0.05. C and D, cells were pretreated with or without actinomycin D (Act D, 0.5 μg/ml) for 30 min, followed by treatment with PACAP (10 nm) for 3 h. mRNA levels of RCAN1 were measured by quantitative real-time PCR (C), and the cell extracts were immunoblotted with anti-RCAN1 and anti-GAPDH antibodies (D). E, characterization of the rat RCAN1.4 gene promoter. F, cells were transfected with the RCAN1.4-luciferase reporter construct (pRCAN1.4-luc). After 24 h, cells were treated with or without PACAP (10 nm) for 20 h and analyzed for luciferase activity. The results were normalized to β-galactosidase activity and are presented as mean ± S.D. of three independent experiments. **, p < 0.01.