Activation of ERK1/2 phosphorylation and MYC expression in sIgD-stimulated CLL and normal B cells. (A) CLL samples were stimulated with anti-IgD for up to 60 minutes and analyzed for ERK1/2 phosphorylation by flow cytometry. Graphs show the fold increase in ERK1/2 phosphorylation after stimulation with anti-IgD relative to untreated cells for 4 representative samples. Note that the same samples are shown in Figure 3A (after sIgM stimulation), and direct comparison of sIgM and sIgD responses is shown in supplemental Figure 1B. (B) Comparison between protracted/transient ERK1/2 responses after stimulation of sIgM (□) or sIgD (■; Fisher exact test, P = .0001; n = 37). (C) Quantitation of MYC protein induction after stimulation of sIgM or sIgD, relative to isotype antibody-treated controls (n = 18). Note values for anti-IgM–treated cells are the same as those shown in Figure 1B and are shown again here to allow direct comparison with anti-IgD–treated cells. See supplemental Figure 1C for side-by-side immunoblot analysis of MYC expression in sIgM- or sIgD-stimulated CLL cells. (D) Normal B cells were stimulated with anti-IgM (□) or anti-IgD (■) for up to 60 minutes and ERK1/2 phosphorylation analyzed by flow cytometry. Graph shows mean fold increase in ERK1/2 phosphorylation for CD20⁺CD27⁻ cells. Data are mean values ± SEM (n = 8). (E) CD19⁺CD27⁻ B cells isolated from healthy donors were stimulated with anti-IgM or anti-IgD for 3 or 6 hours or for 6 hours with the isotype control. MYC and MAX expression was analyzed by immunoblotting. Results are shown for cells isolated from 2 separate donors.