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. 2017 Aug 2;7:7121. doi: 10.1038/s41598-017-07479-z

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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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The bivalent scFvD2-Fc antibody fragment induce FGFR1 dimerization. (a) The bivalent scFvD2-Fc antibody can simultaneously bind two D1 domains of FGFR1. Purified his-tagged, NusA fusion of domain D1 of FGFR1 (NusA-His-D1) was incubated either alone or in the presence of the bivalent scFvD2-Fc or monovalent scFvD2.myc. Next, preformed antibody fragment-NusA-His-D1 complexes were incubated with purified GST fusion of D1 domain of FGFR1 (GST-D1). Proteins bound to the GST-D1 were analyzed by Western blotting using specific antibodies. The interaction of D1 domains with each other is strongly enhanced only in the presence of the bivalent scFvD2-Fc. (b) Oligomeric state of FGFR1 was studied with 2D BN/SDS PAGE. Serum starved NIH3T3 cells were treated with FGF1, scFvD2.myc or scFvD2-Fc. Membrane proteins were solubilized with digitonin and separated in the 1st native dimension using 4–13% BN-PAGE gels. Native gels were sliced and separated in the 2nd denaturing dimension using SDS-PAGE. Oligomeric state of FGFR1 was assessed by Western blotting using FGFR1-specific antibodies. (c) Chemical crosslinking reveals that the bivalent scFvD2-Fc antibody fragment can induce FGFR1 dimerization in cells. Serum starved NIH3T3 cells were incubated with FGF1, scFvD2.myc or scFvD2-Fc and crosslinked with DSS. Cells were lysed, FGFR1 was isolated by co-immunoprecipitation and oligomerization of FGFR1 was assessed by Western blotting using receptor-specific antibodies. Addition of FGF1 strongly induces FGFR1 dimerization. The bivalent scFvD2-Fc also induces FGFR1 dimerization, but to the lower extent than FGF1. In contrast the monovalent scFvD2.myc has no impact on the oligomeric state of FGFR1.