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. 2017 Nov 10;7:15268. doi: 10.1038/s41598-017-15311-x

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Full-length Reelin and Reelin fragment N-R6 interact with L1 and cleave L1 to accelerate migration in vitro. (a) ELISA using substrate-coated L1-Fc, Fc or vehicle solution (phosphate buffer saline, pH 7.3) and purified Reelin protein (RLN^rec), mock-purified solution (mock^rec) or cell culture supernatants from Reelin-expressing (HEK^rln) or Reelin-lacking (HEK^Ø) HEK cells. (b) ELISA using substrate-coated purified Reelin protein (RLN^rec), mock-purified solution (mock^rec) or vehicle solution and L1-Fc or Fc. (a,b) Mean values + SEM for binding are shown (***p < 0.0001; One-way ANOVA with Tukey’s Multiple Comparison Test from 3 independent experiments with triplicates). (c) Pull-down with L1-Fc and Fc using supernatants from Reelin-expressing (HEK^rln) and Reelin-lacking (HEK^Ø) HEK cells. Supernatant from Reelin-expressing HEK cells was used as input control and Reelin antibody G10 was used for detection. A representative immunoblot out of 3 independent experiments with Reelin-positive bands is shown and full-length Reelin (RN-450) and its fragments N-R6 and N-R2 are indicated. (d) Structure of Reelin and its proteolytic fragments after N- and C-terminal processing by ADAMTS-4. The enzymatically decisive serine residue in the GKS₁₂₈₃D sequence in murine Reelin is indicated by a red spot. (e) Lysates from co-transfected HEK cells expressing full-length Reelin (RLN-450) or Reelin fragments N-R6, N-R2, R3-6, R3-8 or R7-8 were subjected to immunoblot analysis with L1 antibody against the intracellular domain (L1-ICD) and GAPDH antibody to control loading. Representative immunoblots out of 6 independently experiment are shown and display all L1 forms and the GAPDH bands. Quantification of full-length L1 (L1-FL) levels (AU: arbitrary units) is shown in the lower panel. Mean values + SEM from 6 independent experiments are shown and differences between groups are indicated (**p < 0.001; One-way ANOVA with Tukey’s Multiple Comparison Test). (f) Images of scratches in monolayers of co-transfected HEK cells expressing L1 and full-length Reelin or one of the indicated Reelin fragments. Gap borders are highlighted with continuous white lines; scale bars: 50 µm. Mean values + SEM from 3 independent experiments with triplicates per group are shown for gap closure after scratching. Differences are indicated (**p < 0.005; One-way ANOVA with Tukey’s Multiple Comparison Test).