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. 2017 Jan 31;10:15. doi: 10.3389/fnmol.2017.00015

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Copyright © 2017 Canu, Pagano, La Rosa, Pellegrino, Ciotti, Mercanti, Moretti, Sposato, Triaca, Petrella, Maruyama, Levi and Calissano.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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TrkA/APP interaction is inhibited by low-affinity NGF receptor p75, ShcC and Mint-2. (A,B) Quantification by FACS analysis of HEK293 cells transfected with the indicated constructs of the % BiFC positive cell and the mean fluorescence intensity. Results are mean ± SEM of three independent transfections performed in triplicate. ^∗∗∗ < 0.0001; compared to TrkA-VC/APP-VN, One way Anova with Bonferroni post hoc test. (C) Western blot analysis showing expression of p75NTR in cell lines indicated. (D) Visualization of TrkA/APP interaction by BiFC analysis in HEK293, NIH-3T3 and HeLa transfected with the indicated plasmids. Fluorescence images were acquired by confocal microscopy 18–24 h after transfection. (E) Half of the cells transfected for FACS analysis were collected, lysed and analyzed by Western blotting with either rabbit anti APP-NT (A8967, Sigma Aldrich), rabbit anti TrkA-NT (H190), rabbit anti p75 NTR polyclonal antibody (N 3908, Sigma Aldrich), mouse anti ShcC, rabbit anti-Mint2 (sc-30135, X11β M-220 Santa Cruz Biotecnology). (F) Cell viability determined by the MTT assay 24 h after transfection. Results are the means (±SEM) of duplicate determinations from three independent experiments and are reported as percentage of mock transfected cells.