Figure 4

Increased Akt activation in NGF-treated PC12-U18666A and in the medial septum of NPC1^-/- mice compared to WT. A. PC12 cells were treated for 24 hrs with 2 μg/ml U18666A and with 5 ng/mL NGF for different durations (0-360 min). After NGF treatment, PC12 cells were homogenized, and the samples were processed for SDS-PAGE, blotted and immunolabeled for phosphorylated Akt (pAKT), total Akt (AKT), phosphorylated ERK1/2 (pERK), total ERK1/2 (ERK), phosphorylated PLC-γ (pPLC-γ) and total PLC-γ (PLC-γ). B. PC12 cells were treated for 24 hrs with 2 μg/ml U18666A (U18666A) or not treated (Control) and then were left untreated (T0) or incubated with NGF for 2 hrs (2 h). After treatment, PC12 cells were homogenized, followed by immunoprecipitation with a polyclonal antibody against Trk (C14, Santa Cruz) and immunoblotting for total Trk (upper panel, monoclonal B3, Santa Cruz) or with a monoclonal antibody against phosphotyrosine (pTyr). The graph shows the quantification of total TrkA in control and PC12-U18666A cells at time 0 (T0) or after 2 hrs of NGF treatment (2 h). The levels of TrkA are equivalent in both experimental groups. C. The graph shows the quantification of pAkt normalized to total Akt ± SEM from three different experiments. The differences between treatments are significant, *p < 0.001 (two-way ANOVA). D. The graph shows the quantification of activated ERK1/2 (pERK) normalized to total ERK ± SEM from five different experiments. The differences between treatments are not significant (two-way ANOVA). E. The graph shows the quantification of activated PLC-γ (pPLC-γ) normalized against total PLC-γ ± SEM from four different experiments. The differences between treatments are significant, **p < 0.05, two-way ANOVA. F. Levels of phosphorylated Akt (pAKT) and total Akt (AKT) were analyzed by western blot of homogenates of the MS of three 8-week-old WT and three 8-week-old NPC1^-/- mice. Bands corresponding to pAkt and Akt from three different WT and NPC1^-/- mice are shown. G. Quantification of the western blot shown in F indicates that pAkt levels are significantly higher in NPC1^-/- mice than in WT mice, *p < 0.01, unpaired Student's t-test. n = 3 animals per phenotype. pAkt levels were normalized against total Akt.