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. 2021 Jun 3;11:11676. doi: 10.1038/s41598-021-91112-7

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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DC-SIGN binding and signaling in WSU-FSCCL cells. (a) Binding of DC-SIGN-Fc or DC-SIGN-HA to WSU-FSCCL and Ramos cells. Cells with no addition were analyzed as a control. Representative of 5 independent experiments. (b) WSU-FSCCL cells were incubated with DC-SIGN-Fc, DC-SIGN-HA, anti-IgM or left untreated as a control for 30 min at 4 °C and then warmed rapidly to 37 °C for the indicated times. Expression of total and phosphorylated AKT, ERK1/2 and GAPDH (loading control) was analyzed by immunoblotting. The gels were run under the same experimental conditions. Molecular weight of proteins markers shown as KDa. Results shown are representative of 3 independent experiments. Full-length blots are shown in Supplementary Fig. 3. (c) Representative iCa²⁺ flux analysis after addition (arrow) of anti-IgM, DC-SIGN-Fc, DC-SIGN-HA or control antibody (control ab) and summary of data for 5 separate experiments. Graph shows mean (± SD) response with values for anti-IgM-treated cells set to 100%. The statistical significance of the indicated differences is shown (paired t-test) (d) Confocal imaging of bound DC-SIGN-Fc (green) and sIgM (red) on fixed WSU-FSCCL cells. Cells were incubated with DC-SIGN-Fc at 4 °C for 1 h and anti-IgM was added for the last 30 min. DAPI staining is shown in blue. Two representative cells shown. (e) Anti-IgM accessibility with DC-SIGN bound to BCR. FACS analysis of WSU-FSCCL cells incubated with DC-SIGN-Fc (left), DC-SIGN-HA (right) or no addition for 30 min at 4 °C and stained with FITC anti-IgM or left unstained as control.