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. 2017 Oct 16;8:947. doi: 10.1038/s41467-017-00983-w

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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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Fig. 1 — FGFR2 binds directly to TRPA1 ankyrin repeats via its C-terminal proline-rich motif. a Representative image of haematoxylin and eosin (H&E) stain and immunohistochemistry (IHC) staining with FGFR2 and TRPA1 antibodies of a lung cancer tissue microarray. Scale bar: 10 μm. Neoplastic epithelial cells are indicated by the red arrow, inflammatory cells by the green arrow, and supporting stroma (fibroblasts) by the black arrow. b Fraction of total LUAD samples (in percentage) in the tissue microarrays (TMAs) with the indicated pathological scores. c Western blots for the expression levels of FGFR2 and TRPA1 in LIMM-NBE1, CCL-204, HCC-515, and HCC-44 cell lines. Normalized densitometric values (in relative arbitrary units) are reported in red below each band and represent the average of three independent experiments. d Duolink assay results with an example of a PLA signal depicted by the white arrow. Scale bar: 50 μm. e Western blot analysis of an immunoprecipitation (IP with anti-TRPA1) in HCC-515 cell line followed by immunoblotting (IB) with the indicated antibodies. f Western blot analysis of an IP in HEK 293T cells transfected with full-length TRPA1 with either full-length FGFR2 (FL) or extracellular region-truncated FGFR2 (ΔE). IP and IB were performed with the indicated antibodies. g GST pull-down results in HEK 293T cells transfected with TRPA1 and C, C4, C10, and C13 GST-constructs on GST beads. h GST pull-down experiment with full-length purified TRPA1 and C, C4, C10, and C13 GST-constructs on GST beads. i Western blot analysis of purified biotinylated full-length TRPA1 or ankyrin repeats-truncated TRPA1 (ΔN) proteins immobilized on streptavidin beads and incubated with full-length purified FGFR2. j Microscale thermophoresis (MST) curve of purified full-length FGFR2 (F2; titrated in a range of concentrations) with purified full-length TRPA1 (A1) or ΔN. Apparent dissociation constant K _d for A1-F2 was 122.8 ± 23.4 nM. Error bars represent the s.d. of each data point (n = 7)