The ΔF508 mutation results in loss of CFTR function and mature protein in native human colon

Characterization of Wild-Type and ΔF508 Cystic Fibrosis Transmembrane Regulator in Human Respiratory Epithelia

“…For protein biochemistry studies, freshly excised nasal and bronchial specimens (ϳ0.25 cm 2 in size) were scraped with a sterile scalpel to collect the superficial epithelial layer, which was rapidly frozen in liquid nitrogen and stored at Ϫ80°C. For Ussing chamber experiments, thin layers of nasal epithelium were dissected under a stereomicroscope (Mall et al, 2004). After Ussing chamber studies, tissues were embedded in OCT and cryo-preserved at Ϫ80°C.…”

“…CFTR mAbs were raised using the full-length human CFTR protein purified from BHK cells (Mall et al, 2004). Rabbit polyclonal antibodies against EBP50 (Mohler et al, 1999), MUC5AC, and InsP3-receptors (Mohler et al, 2004) were gifts from Dr. S. Milgram (UNC-CH), Dr. J Sheehan (UNC-CH), and Dr. P. Mohler (Duke University), respectively; the rabbit polyclonal antibody against AQP5 (Kreda et al, 2001) and the rat monoclonal antibody (mAb) against tubulin were from Chemicon (Temecula, CA); the mAb against ezrin (Mohler et al, 1999) was from BD Transduction Laboratories (San Diego, CA); the mAb against MUC5AC was from NeoMarkers (Fremont, CA); and mAb against ␤IV-tubulin from Sigma (St. Louis, MO); all secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA).…”

“…Thin frozen tissue sections (ϳ8 m) were processed as previously described (Kreda et al, 2001;Mall et al, 2004). Serial tissue sections were incubated at 4°C overnight with CFTR mAbs 528, 769, or a nonimmune mouse IgG (all at 10 g/ml).…”

“…Early immunofluorescence studies described localization of WT but not ⌬F508 CFTR to the apical PM of the sweat duct (Cohn et al, 1991;Kartner et al, 1992) and airway submucosal glands (Engelhardt et al, 1992;Puchelle et al, 1992). Functional studies also failed to detect a cAMP-dependent Cl Ϫ secretion in native CF airway and intestinal epithelia (Boucher et al, 1989;Veeze et al, 1991;Mall et al, 2004). However, the concept that the pathophysiology of ⌬F508 CFTR in native epithelia reflects predominantly defective processing and protein mislocalization has been challenged.…”

“…Next, we characterized whether functional ⌬F508 CFTR reached the PM in airways resected from ⌬F508 homozygous CF subjects with immunocytological and functional approaches and asked whether ERM proteins maintained their apical localization in the presence of persistent CF airways inflammation and infection. This characterization was made possible by the availability of highly sensitive/specific CFTR mAbs (Gentzsch et al, 2003;Mall et al, 2004) and an ample collection of normal and CF airway tissues.…”