P90RSK is synergistically phosphorylated and is involved in neurite outgrowth in all three systems. (a(i)) Time-course of P90RSK phosphorylation at 20 and 60 minutes following NGF (0-50 ng/ml)-PACAP (0-100 ng/ml) treatment. Phosphorylation levels of the proteins were analyzed by Western blotting, and normalized to the levels of Actin. The blots used were the same as those used for pJNK in Figure 2a. (a(ii)), (b(i)), (c(i)) Time-course measurements of pP90RSK at 20 and 60 minutes following NGF (50 ng/ml)-PACAP (100 ng/ml) (NP), FGFb (50 ng/ml)-PACAP (100 ng/ml) (FP) or EGF (50 ng/ml)-PACAP (100 ng/ml) (EP) stimulations, respectively. The total protein levels of P90RSK upon treatment with single ligand or combinations of the growth factors and PACAP were unchanged across all conditions and time-points (Additional file 5: Figure S5). (a(iii)), (b(ii)), (c(ii)) Effect of P90RSK inhibitor, BRD7389 (0.2 μM), on neurite outgrowth in the NP, FP, and EP systems, respectively. (d) Net reduction in neurite outgrowth between combinatorial ligand treatment (bi-ligand) versus the sum of neurite outgrowth reduction from treatment with each ligand separately in the presence of BRD7389 (0.2 μM). Significant differences between the effects of combinatorial experimental treatment of growth factor-PACAP and summation of their individual effects, and that between the effects of treatments with and without inhibitors were calculated using the paired Student’s t-test. A value of p < 0.05 was considered significant (**p < 0.01; *p < 0.05).
Seow et al. BMC Neuroscience 2013 14:153 doi:10.1186/1471-2202-14-153