Figure 6. B-I09 does not affect synthesis, assembly and transport of critical B cell integral membrane proteins.

(A and B) To reveal the effect of B-I09 on membrane-bound IgM (mIgM), XBP-1^WT/μS KO B cells were stimulated with LPS for 2 days and subsequently treated with DMSO (control) or B-I09 (20 μM) for 1 additional day. DMSO-, or B-I09–treated XBP-1^WT/μS KO B cells and DMSO-treated XBP-1^KO/μS KO B cells were radiolabeled for 15 minutes, chased for indicated time, and lysed. Intracellular mIgM and κ light chain were immunoprecipitated from lysates using an anti-κ antibody (A). Secreted free κ chains were immunoprecipitated from culture medium using an anti-κ antibody (B). Immunoprecipitates were analyzed by SDS-PAGE. Data are representative of 3 independent experiments. (C) Lysates similar to those in A were immunoprecipitated using an antibody against the class I MHC heavy chain (HC), and immunoprecipitates were analyzed by SDS-PAGE. Data are representative of 3 independent experiments. (D) Using lysates similar to those in A, we performed immunoprecipitations using an anti-Igβ antibody to retrieve the Igα/Igβ heterodimers. Immunoprecipitated Igα/Igβ proteins were eluted from the beads and treated with endo-H or PNGase F before being analyzed by SDS-PAGE. μM, membrane-bound μ chain; CHO, high mannose-type glycans; CHO*, complex-type glycans; NAG, N-acetylglucosamines. Data are representative of 3 independent experiments.