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. 2017 Jun 12;7:3296. doi: 10.1038/s41598-017-03740-7

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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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DC-SIGN interacted with and bound TLR4. (A) Macrophages were incubated with oxLDL (0, 12.5, 25 and 50 μg/ml) for 6 hours. Cell lysates were immunoprecipitated with a DC-SIGN antibody and probed with an antibody against TLR4. As loading controls, whole cell lysates were probed with antibodies against total TLR4 and α-tubulin. The IgG control had been detected in another Western-blot assay, where the protein level and exposure time were same Western-blot results on the right. (B) Macrophages were stimulated with or without 50 μg/ml oxLDL and stained with DC-SIGN (red) and TLR4 (green). Images were acquired by confocal microscopy (1,200x). Yellow indicates co-localization of the two proteins. (C and D) The maps of pFLAG-CMV-5.1-DC-SIGN and pcDNA3.1 (−)/myc-HisA-TLR4 are shown. pFLAG-CMV-5.1-DC-SIGN or pcDNA3.1 (−)/myc-HisA-TLR4 were transfected into HEK293 cells. The localization patterns of FLAG-DC-SIGN (FLAG: green, DC-SIGN: red) and His-TLR4 (His: green, TLR4: red) were detected by immunofluorescence stains, and the overexpression efficiency of FLAG-DC-SIGN and His-TLR4 were measured by western blot analysis. (E) In vitro pull-down of FLAG-DC-SIGN and His-TLR4 fusion proteins. The total cell lysate (10 μg) of FLAG and FLAG-DC-SIGN were absorbed onto anti-FLAG M2 beads and incubated with the whole cell lysate (10 μg) His-TLR4. Elutes were analyzed by SDS-PAGE followed by immunoblotting with anti-TLR4, anti-His and anti-FLAG. IB: Immunoblotting, IP: Immunoprecipitation.