FIGURE 2.

PACAP regulates MAPK and Akt neurotrophic pathways with distinct temporal profiles. Mature primary sympathetic neuronal cultures (9 days in vitro) were acutely withdrawn from NGF for 4 h before 100 nm PACAP27 replacement for the times shown. The cultures were extracted in the presence of phosphatase/protease inhibitors for SDS-PAGE fractionation and Western blotting analyses as described under “Experimental Procedures.” In separate experiments the order of the different phospho-specific antibody applications to the blots was varied, and the results were identical. Acute NGF withdrawal diminished endogenous ERK1/2, ERK5, and Akt phosphorylation levels, whereas JNK and p38 MAPK activation was enhanced. The PACAP-induced changes in effector phosphorylation demonstrated distinct temporal profiles and in patterns consistent with neuronal survival and abrogation of proapoptotic signals. Blots were hybridized for actin immunoreactivity as a final step to demonstrate near equal sample loading. Data are representative of three independent experiments.