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Fig. 5. Mutagenesis and enzymatic deglycosylation of individual CFTR N-glycosylation sites. (A) Western blot of CFTR variants N-glycosylated at both, either, or neither of the two native sites. 1% SDS lysates of stably expressing CHO cells were resolved on 6% polyacrylamide gels, transferred to nitrocellulose and probed with mouse monoclonal antibody M3A7. (B) Endoglycosidase H treatment of CFTR individual glycosylation site mutants. Total cell lysates were incubated with (+) or without (–) endoglycosidase H at 37°C for 4 hours and analyzed by western blotting. The arrowheads indicate core-glycosylated protein or protein deglycosylated by endoglycosidase H. (C) Individual glycosylation site variants have different sensitivities towards deglycosylation by N-glycanase. Lysates of stably expressing CHO cells were incubated with 1 U N-glycosidase F at 37°C for 0 (lane 1), 10 minutes (lane 2), 20 minutes (lane 3) or 24 hours (lane 4) and subjected to SDS-PAGE and western blotting.
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