Fig. 2.

CD40 stimulation enhances Cbl-b ubiquitination induced by IgM stimulation in B cells. A. Splenic B cells (5 × 10⁶) from BALB/C mice were stimulated for 5 min with different concentrations of F(ab’)₂anti-IgM (2 μg/ml) and anti-CD40 (2 μg/ml), or both, and lysed in RIPA buffer. The cell lysates were immunoprecipitated with anti-Cbl-b, and blotted with anti-Ub mAb. B. Splenic B cells (5 × 10⁶) from WT and Cd40^−/− mice were stimulated for 5, 15, 30, 60, and 120 min with F(ab’)₂anti-IgM (2 μg/ml) and anti-CD40 (2 μg/ml), and lysed. Cbl-b expression was determined by immunoblotting. C. WT B cells were pre-treated for 1 h with lactacystin (20 μM), stimulated for 0, 15 and 30 min with F(ab’)₂anti-IgM and anti-CD40, and lysed. Cell lysates were blotted with anti-Cbl-b mAb. These data are representative of three independent experiments.