Fig. 5

Glycosylation of MPL is essential for the activatory mechanism of CALR mutants, which are strongly secreted. a Empty vector (EV), WT CALR, del52, and ins5 expressing 32D MPL cells were treated with tunicamycin (T; 10 μg/ml), brefeldin A (B; 5 μg/ml), or the solvent methanol (Me) as control. Lysate preparation, SDS-PAGE, and Western blotting were performed. Indicated antibodies were used to confirm downstream signaling and CALR expression. GAPDH staining served as loading control. b Concentrated supernatants (30 μg protein) of indicated 32D cells, which were treated for 6 h with 5 μg/ml BFA or left untreated, were applied to SDS-PAGE followed by Western blotting and compared to the appropriate cellular lysates (30 μg protein). c 32D MPL cells expressing C-terminally YFP-tagged WT CALR, del52, or ins5 were FACS sorted for equal YFP-intensities and YFP signal was monitored until 10 days after sorting. Strong decrease of CALR del52-flag-YFP and ins5-flag-YFP could be observed