Figure 5.
BCR-dependent activation of the canonical NF-κB pathway is impaired in CIN85 bKO B cells. (A) Purified spleen B cells were stimulated with 10 µg/ml anti-IgM F(ab’)2 for indicated times (minutes). The cells were lysed and subjected to SDS-PAGE. Transferred membranes were probed with the indicated antibodies. (B) Spleen B cells from control or CIN85 bKO mice were stimulated with 10 µg/ml anti-IgM F(ab’)2 fragment for the indicated times. After stimulation, some of the cells were subjected to SDS-PAGE to measure the phosphorylation status of IκBα (top). Other cells were collected, precipitated by anti–IKK-γ antibody and subjected to an IKK kinase assay using GST-IκBα as a substrate. The amount of phosphorylated GST-IκBα was detected by Western blotting as described in the Materials and methods (bottom). Phosphorylation status of IKK-β was also analyzed by Western blotting. p-ERK was examined with whole cell lysate. Representative data of at least three independent experiments are shown.