Figure 4
From: Rewiring of sIgM-Mediated Intracellular Signaling through the CD180 Toll-like Receptor
Modulation of sIgM-mediated signaling by sensitization with anti-CD180 mAb. (A–C) Control B cells or CD180+sIgM+CLL samples were treated with anti-IgM F(ab)2 alone or pretreated with anti-CD180 followed with anti-IgM F(ab)2 as described in the Materials and Methods. Unstimulated cultures (Medium) were used as controls. The cells were stained with anti-CD19, fixed, permeabilized and stained with antibodies to p-AKT and p-p38MAPK. P values were calculated using the paired t test. (D) Representative immunoblots of p-AKT, p-PI3K, p-p38MAPK, Mcl-1 and Bc-xL following sequential ligation of CD180 followed by sIgM. Bcl-2 was used as a loading control. The bands were assessed and visualized as described in the Materials and Methods. (E) Relative optical density, normalized to Bcl-2, for p-AKT, p-PI3K, p-p38MAPK, Mcl-1 and Bcl-xL immunoblots, n = 5. (F) The percentages of DiOC6dim (apoptotic) cells in unstimulated CLL cells (Medium) or stimulated with anti-IgM F(ab)2 alone or first with anti-CD180 followed by anti-IgM F(ab)2 for 24 h, stained with anti-CD19 mAb, loaded with DiOC6 and analyzed by flow cytometry. The values represent mean ± SD, p value calculated using the nonparametrical U test.