Figure 3
From: Rewiring of sIgM-Mediated Intracellular Signaling through the CD180 Toll-like Receptor
Anti-IgM mediated phosphorylation of AKT, p38MAPK, BTK and PI3K, expression of Mcl-1, Bcl-xL and apoptosis in AKT-S and p38MAPK-S CLL cells. (A, B, C) Control B cells or AKT-S and p38MAPK-S CLL samples were incubated with anti-IgM F(ab)2 for 10 min (anti-IgM) or left unstimulated in medium (Medium), washed, stained with anti-CD19 mAb, fixed, permeabilized and stained with anti-p-AKT, anti-p-p38MAPK and anti-p-BTK mAbs as described in the Materials and Methods. The results were analyzed by flow cytometry and expressed as percentages of positive cells. P values were calculated using the paired t test. (D) Representative immunoblots show the levels of total PI3K, p-PI3K, Mcl-1 and Bcl-xL in AKT-S and p38MAPK-S CLL samples in unstimulated cultures (Medium) and following stimulation with anti-IgM F(ab)2 as described in the Materials and Methods. Bcl-2 was used as a loading control. (E) Relative optical density, normalized to Bcl-2, for p-PI3K, Mcl-1 and Bcl-xL immunoblots, n = 5. (F) The percentages of DiOC6dim (apoptotic) cells in control B cells, AKT-S, p38MAPK-S and DS CLL samples measured following stimulation with anti-IgM F(ab)2 for 24 h, compared with unstimulated cultures (Medium), as described in the Materials and Methods. The values are mean ± SD, p values were calculated using a nonparametrical U test.