Oligosaccharide side chains of processing forms for sTNFR1-tm and sTNFR1-tm-Y. (A) RBL cells transfected with sTNFR1-tm were biosynthetically radiolabeled for 60 minutes (pulse) and chased for 3 hours before analysis of cell extracts. Due to the slow secretion, the cell supernatant was obtained after a 15-hour chase to have enough secreted sTNFR1-tm for analysis. After pulse and chase, 20 × 106 cells were withdrawn, and after lysis, subjected to immunoprecipitation with anti-sTNFR1. Aliquoted immunoprecipitates were incubated either with Endoglycosidase-H (Endo-H) or N-glycosidase F (N-Glyc), or remained untreated as control, and analyzed by SDS-PAGE. The pulse cell lysate showed partial Endo-H sensitivity, while the chase cell lysate showed total Endo-H resistance. The cell supernatant showed Endo-H resistance. The position of sTNFR1-tm is indicated with an arrow. Molecular weight markers are shown to the left in kDa. (B) Similar experiments were performed with RBL cells transfected with sTNFR1-tm-Y. Cells were biosynthetically radiolabeled for 45 minutes (pulse) and chased for 3 hours before analysis of cell extracts. The pulse sample showed partial Endo-H sensitivity, while the chase sample showed total Endo-H resistance. Secretion was too low to yield enough material for analysis.
