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. 2019 Nov 7;9:16272. doi: 10.1038/s41598-019-52655-y

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© The Author(s) 2019

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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Fluorescent A555-TSP-4 protein does not bind to membrane-bound α₂δ-1 in HEK293-EBNA cells. (A) Representative confocal images of the immunocytochemical detection of membrane-localised α₂δ-1 FL NTST over-expressed in HEK293-EBNA cells. Cells were transfected with either empty vector (right column) or vector encoding full-length α₂δ-1 containing an N-terminal strep II-tag (α₂δ-1 FL NTST, left column). Cells were stained either without permeabilisation (upper row) or after membrane permeabilisation (lower row). Signals of WGA conjugated with Alexa Fluor 633 (green) and α₂δ-1 (red) are shown individually in the small images; merged signals are shown in the large images. DAPI was used to visualise the nucleus (blue). Images show top view as well as upper-side (green box) and right-side (red box) views of a single slice of scanning near the middle of cells. Scale bar is 20 µm for all images. (B) Binding of A555-TSP-4 or TSP-4 to α₂δ-1_S NTST analysed by an ELISA-style ligand binding assay. α₂δ-1_S NTST (20 µg/ml) was coated onto 96-well plates and incubated with either TSP-4 or A555-TSP-4 at two different concentrations (250 and 500 nM) for each protein. Specific binding was calculated by subtracting OD values of non-specific binding from those of total binding. Data represent mean ± SEM of 2 independent measurements, each performed in duplicate. OD values for specific binding of TSP-4 and A555-TSP-4 were subjected to unpaired Student’s t-test. No significant difference was found (P > 0.05) for each of the two concentrations of both TSP-4 species used. (C) Cells transfected with either empty vector or vector encoding α₂δ-1 FL NTST were incubated with increasing concentrations of A555-TSP-4 (final concentration: 9–909 nM) in 96-well imaging plates. Control wells received the same volume of dilution medium (40 µl) without A555-TSP-4. Average A555-TSP-4 fluorescent signal intensities from wells containing cells transfected with α₂δ-1 FL NTST encoding vector or empty vector (±SEM of 3 independent experiments, each performed in triplicate) were plotted versus the A555-TSP-4 concentration.