Fig. 4. Transport of ERGIC-53-based reporters. The luminal domain (L53) derived from ERGIC-53 carries a c-myc tag and an N-glycosylation site. L53 constructs with an ERGIC-53 wild-type TMD are designated L53T53 and constructs with an 18 leucine TMD L53L₁₈. (A) Substitution of the TMD and cytoplasmic domain of ERGIC-53 does not interfere with oligomerization. 48 hours after transfection, the cells were labeled for 5 minutes with [³⁵S]methionine and chased for 15 minutes. Cells were washed and lysed in the presence of 20 mM iodoacetamide before immunoprecipitation with anti-myc. Immunoprecipitates were separated by 4-10% gradient SDS-PAGE under reducing (+DTT) or non-reducing (-DTT) conditions followed by fluorography. M, M_r marker lane. (B) Transport efficiency of constructs probed by pulse-chase/endo H (Fig. 1). This analysis also included monomeric forms of constructs L53L₁₈R₂S₁₀ and L53L₁₈R₂S₉V denoted mL53L₁₈R₂S₁₀ and mL53L₁₈R₂S₉V. The white and black bars represent the same controls as in Fig. 1. Means±s.e.m. of at least three independent experiments.