FIGURE 6.

Modulation of APP/TrkA association by NGF and trafficking perturbation. (A) Representative images of PLA performed with APP-CT and Trk B3, antibodies showing the interaction of APP and TrkA in primary septal cultures (12 DIV) untreated (Ctrl) or treated with NGF (100 ng/ml for 1 h). Positive signal of interaction is shown as red dot, nuclei are stained with DAPI. (B) Quantification of the PLA signal in primary neurons untreated and treated with NGF (100 ng/ml for 1 h)). The graph represents quantitative analysis of the number of PLA fluorescent red puncta/cell counted in five different fields and for a total of 20–30 cells examined and are expressed as mean ± SEM ^∗∗p < 0.01 Student t-test. (C) Co-localization of PLA signal with GM130 and calnexin in untreated and NGF treated cells. A representative z-stack of images was collected from each cell. (D) Confocal microscopy images of immunofluorescence and PLA signal [both performed with anti-APP-CT (red) and anti-Trk B3 (green) antibodies], of untreated (Ctrl) and BFA (5 μg/ml for 3 h), cytochalasin D (5 μM for 3 h) and colchicine (100 μM for 3 h) treated primary septal cultures (12 DIV) are shown. PLA dots in cytocalasin D treated cells are distributed in cell body and also along the neuritis coherently with the co-localization signal detected by immunofluorescence. Nuclei are stained with DAPI (blue). (E) Quantification of the PLA signal of APP/TrkA interaction in primary neurons untreated (Ctrl) and treated with BFA, cytochalasin D and colchicine. A total of 20–30 cells were examined. ^∗∗∗p < 0.001 when compared to Ctrl. Student t-test was used for statistical analysis. (F) Representative Western blot analysis with anti APP-CT (A8717, Sigma Aldrich), anti TrkA-CT (Abcam, ab76291) antibodies and anti β-actin as loading control under treatments indicated.