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. 2016 Dec 15;9:143. doi: 10.3389/fnmol.2016.00143

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Copyright © 2016 Zuko, Oguro-Ando, Post, Taggenbrock, van Dijk, Altelaar, Heck, Petrenko, van der Zwaag, Shimoda, Pasterkamp and Burbach.

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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Interaction of Cntn6 and Lphn1 in vitro and in vivo. (A) The architecture of the native Cntn6 protein and the structures of the Cntn6 and control fusion proteins tagged with GFP and biotin. (B) Expression of Cntn6-TMGFPBio and TMGFPBio in HEK293 cells, detected by fluorescence (green), anti-Cntn6 antibody (red), and DAPI (blue) staining. Scale bar represents 30 μm. (C) Precipitations were performed by anti-GFP-coupled beads and eluates from anti-GFP-coupled beads were analyzed on Western blot using an anti-GFP antibody. (D) Coomassie blue stained the NuPage 4–12% gels, which were submitted to mass spectrometry analysis. Red dots indicate respective expressed fusion proteins. (E–H) HEK293 cells were cotransfected with Cntn6 and Lphn1 expression plasmids tagged with either GFP or HA. After IP of the GFP-tagged protein by anti-GFP antibodies the eluates were analyzed by Western blot. (E) Immunoblotting with anti-HA and anti-GFP antibodies revealed HA-Lphn1 coprecipitation with Cntn6-TMGFPBio, (F) but not with control GFP. (G) HA-Cntn6 was coprecipitated with Lphn1-GFP, (H) but not with control GFP. (I) Proteins were IPed from wild-type P14 mouse cortex lysates using an anti-Cntn6 antibody. Blots stained with antibodies against Cntn6 and both p85- and p120-fragments of Lphn1 revealed interaction between Cntn6 and Lphn1. No coprecipitation was found in normal IgG control IP. Molecular weights are as follows: Cntn6-TMGFPBio = 147.8 kDa; TMGFPBio = 39.6 kDA; Lphn1-GFP = 125 kDa; GFP = 27 kDa; HA-Cntn6 = 141 kDa; HA-Lphn1 = 131 kDa; Cntn6 = 130 kDa; Lphn1-p85 = 85 kDa; Lphn1-p120 = 120 kDa. Ig-like, immunoglobulin-like; FNIII, fibronectin type III; TM, transmembrane domain; GFP, green fluorescent protein; Bio, biotin.