FIG. 8.
SHIP-1 is required for JNK regulation in ex vivo mouse B cells. B cells were purified from SHIP-1+/+ and SHIP-1−/− mice and then propagated for 60 h in LPS-containing culture medium as detailed in Materials and Methods. (A) Flow cytometry. After propagation in LPS-containing medium, B-cell populations were identified by flow cytometry using antibodies against surface IgM and surface IgD. Percentages of cells in the various populations are shown. (B) BCR-induced proliferation. LPS-propagated cells were stimulated for 48 h with the indicated concentrations of F(ab′)2 fragments of GAM IgM. Proliferation was then assessed by measuring tritiated thymidine incorporation. Assays were done in triplicate. Average values with standard deviations are shown. (C) LPS-induced proliferation. The experiment was conducted as detailed for panel B, except that cells were stimulated with LPS (20 μg/ml). Assays were done in triplicate, and average values with standard deviations are shown. (D) BCR-triggered activation of MAPKs. Cells were stimulated and lysates were analyzed as detailed for Fig. 7. (E) Levels of MAPKs in ex vivo mouse B cells. The abundance of the various MAPKs in the cells used in panel D was assessed by immunoblotting of cell lysates with the indicated antibodies.