Fig. 7. Mannose trimming is required for µCH1 condensation and efficient sRB formation. (A,B) Kifunensine causes accumulation of µCH1 but no sRB formation in myeloma transfectants. N[µCH1] were treated for 40 hours with sodium butyrate (NaBut, 1 mM) or with kifunensine (Kif, 2 µg/ml), to increase the expression or prevent the degradation of µCH1, respectively. Cells were then fixed and processed for immunofluorescence microscopy with anti-µ (A, bar 5 µm) or lysed in TX100. Equal amounts of soluble (s) and insoluble (i) lysate fractions were analyzed by western blotting (B). (C,D) Kifunensine causes accumulation of detergent-soluble µCH1 in the ER of HeLa cells. Cells were cultured for 5 days without tetracycline, with or without 2 µg/ml kifunensine, and processed for immunofluorescence microscopy (C, bar 5 µm) or western blotting (D). Kifunensine was re-added every 24 hours (Tokunaga et al., 2000). In both lymphoid and non-lymphoid cells, kifunensine caused accumulation of abundant µCH1, which was mainly in the soluble fraction when mannose trimming was prevented. As expected, µCH1 chains present in kifunensine-treated cells display slower electrophoretic mobility. (E,F) The association between ERGIC-53 and µCH1 is inhibited by kifunensine. H[µCH1] cells were cultured in the absence of tetracycline for 2 days, with or without kifunensine (Kif, 2 µg/ml, lanes 5-6) and then transfected with ERGIC-53 wt. After further 40 hours of culture, cells were treated with 50 µM CPA for 2 hours (lanes 3-4) before co-immunoprecipitation and quantification, as described in the legend to Fig. 6C. Kifunensine was present for the entire duration of the experiment.