Fig 1: 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 ß-actin as loading control under treatments indicated.
Fig 2: Identification of the APP and TrkA regions involved in APP/TrkA interaction. (A) Schematic diagram of APP constructs used in this study. All constructs are derived from the human APP-695 isoform. This transmembrane glycoprotein consists of 695 amino acids and can be divided into an extracellular region, a transmembrane domain (TMD) and a small intracellular domain (ID) the extracellular regions is divided into the E1 and E2 domains, linked by an acidic domain. The domain E1 consists of a growth factor-like domain (GFLD) and the following copper-binding domain (CuBD). (B) Cell lysates of HEK293 cells, transfected with the indicated plasmids, were immunoprecipitated with mab Trk (B3) or mab Bcl2 (C-2) (IgG) as control and probed with rabbit anti C-terminal (CT), N-terminal (NT) APP antibodies and rabbit TrkA-CT (Abcam ab76291). Total protein extract (30 µg unbound) (INPUT) was also loaded as positive internal control of electrophoretic mobility and immunoprecipitation efficiency. Each co-transfection has been performed at least three times with similar results. (C) Schematic diagram of TrkA constructs used in this study. All constructs are derived from human TrkA. This receptor consists of 796 amino acids and can be divided in the extracellular ligand-binding domain, [comprising leucine-rich motifs (LRR), two cysteine clusters (C1 and C2) and two immunoglobulin (Ig)-like domains], the TMD, and the intracellular tyrosine kinase domain. (D) Cell lysates of HEK293 cells transfected with APP-695 and TrkA constructs were immunoprecipitated with mab 22C11 or mab Bcl2 (C-2) (IgG) as control and then probed with rabbit anti-TrkA-CT (Abcam, ab76291) and TrkA-NT (H-190 (sc-14024). INPUT corresponds to 30 µg of total unbound extract as in (B) * and ** indicates the IgG heavy and light chains respectively. (E) Immunoprecipitation with anti-TrkB3 from lysate of HEK293 cells, transfected with APP-695 and TrkA constructs, in the presence or absence of 5 mM EDTA. INPUT corresponds to 30 µg of total unbound extract as in (B). Arrow and arrowhead indicate the partially glycosylated and fully glycosylated forms of TrkA, and the immature and mature forms of full length APP.
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