Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2020 Sep 29;218(1):e20200815. doi: 10.1084/jem.20200815

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2020 Nagata et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

Figure 1. — Identification of αCAG as the Mincle ligand in H. pylori. (A, C, F, G, I, and O) Lipid extract from H. pylori was analyzed by HPTLC using chloroform/methanol/water (65:25:4, vol/vol/vol) and stained with copper(II) acetate-phosphoric acid (A, C, F, I, and O) or orcinol (G). Open and closed arrowheads denote the origin and the solvent front, respectively. (B) BMDCs from WT, Myd88^−/−, Card9^−/−, and Fcer1g^−/− mice were stimulated with HPTLC-separated fractions from H. pylori lipid extract for 1 d. The concentrations of TNF in the supernatants were determined by ELISA. (D) NFAT-GFP reporter cells expressing mouse (hereafter omitted for clarity unless needed) Mincle + FcRγ, MCL + FcRγ, Dectin-2 + FcRγ, or FcRγ alone were stimulated with HPTLC-separated fractions from H. pylori lipid extract for 20 h. Induction of GFP was analyzed by flow cytometry. (E) Mincle-CD3ζ– or MCL-CD3ζ–expressing NFAT-GFP reporter cells were stimulated by individual HPTLC-separated fractions of H. pylori lipid extract for 20 h. Induction of GFP was analyzed by flow cytometry. (H) BMDCs from WT and Clec4e^−/− mice were stimulated with HPTLC-separated lipids from H. pylori lipid extract for 1 d. The concentrations of TNF in the supernatants were determined by ELISA. (I) Fraction 13-14 was purified by HPTLC using chloroform/methanol/water (65:25:4, vol/vol/vol) and visualized by copper(II) acetate-phosphoric acid staining. (J) Reporter cells expressing Mincle + FcRγ or FcRγ alone were stimulated by fraction 13-14 (0.01, 0.1 and 1 µg/well) for 20 h and analyzed for GFP expression. (K) Human Ig or Mincle-Ig was incubated with plate-coated fraction 13-14 (0.01, 0.1, and 1 µg/well). Bound proteins were detected with anti-human IgG-HRP. (L) Full-scan MS spectra of fraction 13-14 in the positive ion mode. Ion peak at m/z 781.5979 [M + Na]⁺ is assigned as cholesteryl acyl-pyranoside (calculated mass, 781.5953). (M) ¹H-NMR (600 MHz, CDCl₃:CD₃OD [1:1], 298 K) spectrum of fraction 13-14. (N) Chemical structure and ¹H-NMR chemical shifts assignment of fraction 13-14. Chemical shifts are given in δ ppm, followed by integration, multiplicity, and J in hertz. (O) Reporter cells expressing Mincle + FcRγ were stimulated by HPTLC-separated lipids from WT (H. pylori^WT) or Hp0421-deficient H. pylori (H. pylori^Δ^hp0421) for 20 h and analyzed for GFP expression. The black arrow indicates αCAG, and the gray arrow indicates cholesterol (Chol). (P) WT BMDCs were stimulated for 1 d with lipid subfractions from H. pylori^WT or H. pylori^Δ^hp0421 as in O. The concentrations of TNF in the supernatants were determined by ELISA. Data are presented as the mean ± SD of triplicate (B, H, K, and P) or duplicate (D, E, J, and O) assays and representative of two (B, D, E, H, K, O, and P) or four (J) independent experiments.