Figure 6.

Effect of PACAP treatment on BDNF expression in the mouse brain and in human SK-N-MC PAC1 cells. Animals were treated as described in Fig. 3. A) Quantification of the BDNF mRNA level by quantitative real-time RT-PCR (n=7 animals/group). Quantification of mRNA levels was performed as described in Fig. 3. B) Representative Western blot and quantitative analysis of pro-BDNF expression; 100 μg of mouse brain membrane proteins was analyzed (n=12 animals/group). C) Quantification of soluble BDNF detected in mouse brain supernatants by sandwich ELISA (Promega). Values are expressed as mean ± sd percentage of values from control mice (n=7 animals/group). D) PACAP-induced CREB phosphorylation in SK-N-MC PAC1 cells. Cells were incubated for 4 h with 300 nM PACAP27 in DMEM (lanes 2, 4) or only in DMEM (lanes 1, 3), and then cells were incubated for 24 h in the absence (lanes 1, 2) or presence of 10 μM Aβ₄₂-oligomeres (lanes 3, 4). Cells were collected, and analysis of phospho-CREB and CREB proteins in cell lysates was performed as described in Materials and Methods. E) Influence of PACAP treatment on BDNF mRNA expression in SK-N-MC PAC1 cells. Cells were treated as described above, then RNA isolation end quantitative real-time RT-PCR was performed as described in Materials and Methods. *P < 0.05, **P < 0.01, ***P < 0.001; unpaired Student's t test or 1-way ANOVA/Bonferroni post hoc test.