Immunoblot analysis of CFTR expression in nasal polyps and the intestine of non-CF and ΔF508 homozygous CF patients. Discrimination of CFTR glycoforms by deglycosylation (generation of unglycosylated band A) with N-glycosidase F and endoglycosidase H; the latter reacts with core-glycosylated CFTR (band B) but not with mature CFTR (band C). Deglycosylations were for 3 hours at 37°C in the presence of protease inhibitors. (a) The major CFTR immunoreactive bands of non-CF and ΔF508 homozygous CF nasal polyps and intestinal tissue specimens were indistinguishable from complex-glycosylated band C of T84 cells. In the intestine, CFTR expression was highest in membrane preparations from duodenum and decreased in tissue specimens from jejunum and ileum. Immunodetection was performed with MATG1104 (1:500). (b) Complex modification of the major CFTR immunoreactive signal of ΔF508 homozygous CF intestine (right) is demonstrated by sensitivity to N-glycosidase F and resistance to endoglycosidase H, as is the case for mature CFTR of control non-CF intestine (middle) and T84 cells (left). Immunodetection with M3A7 (1:500). (c) Specificity of the immunoreactive bands from intestinal tissue specimens for CFTR is demonstrated by the characteristic mobility shifts in deglycosylation assays with N-glycosidase F and endoglycosidase H and by peptide competition for antibody binding with the CFTR immunogen. (1) From adult non-CF duodenum; (2) from jejunum of the same patient; (3) from adult ileum of a ΔF508 homozygous CF patient. “a” lanes show deglycosylation of the membrane preparations with N-glycosidase F. “b” lanes show peptide competition with the CFTR immunogen (1 μg MATG1104 preincubated at 20°C with 5 μg CFTR peptide 722–734). Immunodetection with MATG1104 (1:500).