Robust activation, Ab production, and plasma cell differentiation of HSP90b1 KO B cells in vitro. (A) BCR ligation triggers similar level of phosphorylation of downstream signaling molecules in WT and KO B cells. Splenic B cells from WT and KO mice were stimulated with anti-μ F(ab′)₂ for 0 to 30 minutes, followed by intracellular staining and flow cytometric analysis of phosphorylation of Erk, p38, and Syk in B220⁺ populations. Shaded histograms represent unstimulated cells (time 0); open histograms indicate results after stimulation. (B) Purified WT and HSP90b1 KO B cells were labeled with CFSE, stimulated with CD40 Ab and IL-4 for 5 days, and analyzed by flow cytometry for cell surface CD44 and various Ig subclasses. Undivided (CFSE^high) cells were indicated. Numbers represent the percentage of cells in each quadrant over the total number of cells. (C) Same as in panel B, except that cells were stimulated with media or IL-4 and CD40 for 4 days before analysis for plasma cell marker CD138. (D) Splenic CD19⁺ B cells were stimulated with IL-4 and agonistic CD40 Ab for 3 days, then metabolically labeled with ³⁵S-Met/Cys for 30 minutes, and then chased with a saturated amount of unlabeled Met/Cys for 30 to 180 minutes. Ig was isolated by immunoprecipitation (IP) with Ab against κ chain, resolved on reducing SDS-PAGE, and visualized by autoradiography. (E) Same as in panel D, except that cells were metabolically labeled with ³⁵S-Met/Cys for 4 hours followed immediately by IP with protein G (PG) or anti-κ Ab plus PG from both cell lysates and supernatant.