Fig. 4. Gathering of integrins α5/β1, Lewis^b, and sialyl Lewis^x by CAG or CAG-containing OMV in the rafts region.

a Integrins α5 and β1 and b Integrin β1 gathered at the rafts region by treatment of AGS cells with CAG (a) or with H. pylori 26695 OMVs (b). AGS cells were treated with CAG(18:0) for 1 h (a) or with OMVs for 8 h (b), and then fixed. Immunofluorescent staining was then applied to indicate lipid rafts (red), integrin α5 or β1 (cyan), and nuclei (blue). The co-localization of the first two is shown in color of fair pink. Scale bars: 20 (upper) and 5 (lower) μm. c–e Effects of integrin-blocking antibodies on H. pylori adhesion, CagA translocation, and the related tyrosine phosphorylation. AGS cells were treated with or without the integrin α5- or β1-blocking antibodies for 1 h, and incubated in the presence or absence of CAG(18:0) for additional 1 h, and then infected with H. pylori 26695. Adherence was measured by flow cytometry analysis (c) as the proportion of adhered cells with H. pylori (%; shown in each plot). The quantitation made in c was summarized in (d). Levels of CagA translocation and CagA tyrosine phosphorylation were detected by immunoblotting and shown in e. f, g Gathering of Lewis^b (f) and sialyl Lewis^x (g) in the rafts region by treatment of KATO III cells with H. pylori 26695 OMVs. The procedure and staining were similar to those of (b). Scale bars: 20 (upper) and 5 (lower) μm. h and i stand for quantitation of the confocal images made in f and g, respectively. j, k Effect of Lewis^b-blocking antibodies on H. pylori adhesion. The procedure was similar to that of c and d. In d, h, i, and k, data are provided in Supplementary Data 1 and shown as mean ± SD and all statistically significant differences are indicated with asterisks; ***p < 0.001, **p < 0.01, *p < 0.05 vs. the control group (n = 3). Uncropped immunoblot images for e are provided in Supplementary Fig. 4.