SLO injury of the PM reduces BCR activation and internalization. (A–D) BCR–CTB coclustering in the presence or absence of SLO. B cells were incubated at 4°C with Cy3-Fab (5 µg/ml; −XL) or AF546-F(ab′)2 anti–mouse IgM+IgG (5 µg/ml; +XL) plus AF488-CTB (3 µg/ml) and warmed to 37°C for 5 min with or without SLO, followed by fixation and confocal microscopy. (A) Representative images. Bar, 2.5 µm. (B) Percentages (± SD) of all B cells exhibiting polarized BCR clusters (capping). (C) Percentages of B cells with polarized BCR clusters showing impaired BCR caps. (D) Percentages of B cells with polarized BCR clusters showing BCR–CTB coclustering. Data in B–D were generated by visual inspection of images from four independent experiments. (E and F) Tyrosine phosphorylation (pY) of B cells treated with or without SLO. B cells were incubated at 4°C with SLO and AF546-F(ab′)2-goat anti–mouse IgM+IgG (5 µg/ml) and warmed to 37°C for 5 min, followed by fixation, permeabilization, and staining for phosphotyrosine (pY) and analysis of confocal microscopy (E) and flow cytometry (F). Shown are representative images and the mean MFI (± SD) of pY from three independent experiments. Bar, 2.5 µm. (G) Effect of SLO treatment on BCR internalization. B cells were incubated at 4°C with biotinylated F(ab′)2 anti–mouse IgM+IgG (10 µg/ml), followed by 37°C incubation with or without SLO for the indicated times. Cells were then labeled with PE-streptavidin at 4°C and analyzed by flow cytometry to determine the percentage of surface-labeled BCRs remaining on the cell surface. Shown is the mean percentage (± SD) from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P < 0.0001.
