Protein structure of CFTR and target regions of anti-pCFTR antibodies. Two different epitopes (stars) were selected for generating anti-pCFTR antibodies pCFTR-N (corresponding to aa 410–423, EKAKQNNNSRKISN) and pCFTR-C (corresponding to aa 1451–1464, LLPHRNSSKQRSRS). pCFTR, porcine cystic fibrosis transmembrane conductance regulator; NBD, nucleotide-binding domain; R, regulatory domain; MSD, membrane-spanning domain. Drawn using a description from the CFTR database.

Broad tissue expression of pCFTR mRNA detected by RT-PCR. (A) Strong expression of pCFTR mRNA was detected in the upper airways (Lanes 1–7), the heart (Lane 8), the glandular part of the stomach (Lane 11), all segments of the intestine (Lanes 12–17), and the gall bladder (Lane 21). pCFTR mRNA expression was also strongly detected in the uterus, testicle, and epididymis (not shown) and weakly detected in the parotid gland (Lane 9), the esophagus (not shown), the non-glandular part of the stomach (Lane 10), the urinary bladder (Lane 20), and the pancreas (not shown). All other organs were negative, including the brain, the kidney, and all three regions of the skin. A fragment of the mRNA coding for the housekeeping gene EF-1a was RT-PCR–amplified to control for RNA integrity and efficacy of reverse transcription. The RT-PCR results shown are representative for all animals examined. Lane 22: No template control. (B) Laser capture microdissection of bronchial epithelial cells (left panel) and submucosal glands (right panel). Cells are shown before (upper panel) and after (lower panel) dissection. Bar = 50 μm. (C) pCFTR mRNA was consistently detected by RT-PCR in bronchial epithelial cells and submucosal gland cells.

pCFTR is located apically to the Golgi apparatus and occasionally in the apical cell membrane. Using immunofluorescent detection on formalin-fixed, paraffin-embedded tissues, the pCFTR protein was detected in the cytosol of crypt epithelial cells, including goblet cells and non-goblet cells. In the majority of cells, the pCFTR protein (red) was detected apically to the Golgi marker 58K (green). Punctate signals were occasionally detected in or directly beneath the apical plasma membrane (arrows). Left panel, crypt epithelial cells in the colon; right panel, higher magnification of single cells; yellow dots, apical cell membranes; asterisks, locations of the nuclei. Antibody pCFTR-C-1, 1:100 dilution. Bar = 20 μm.

Phylogeny of cystic fibrosis transmembrane conductance regulator (CFTR) orthologs in closely related mammals. The tree was based on a genetic distance tree with branches confirmed by the most parsimony method shown in bold and branch nodes occurring more than 60 times in 100 neighbor joining trees indicated. Ten species of fish, amphibians, birds, and non-eutherian mammals were defined as outgroup.

Anti-pCFTR antibodies detected an ∼170-kDa protein on immunoblots of transiently transfected HEK 293 cells. Antibody pCFTR-C-1 detected a specific band between 150 and 170 kDa (asterisks). This band was not detected in lysates from vector-alone-transfected cells (mock-transfected) or when the respective preimmune serum was used (pCFTR-C-1 pre), and the band disappeared when the antibody was preincubated with the respective peptide (pCFTR-C-1 preinc.). Immunopurified antibody pCFTR-C-1/2-p detected a similar protein between 150 and 170 kDa. All immune sera were diluted 1:1000.

The pCFTR protein is expressed in several epithelial and glandular tissues. The pCFTR protein was detected on formalin-fixed, paraffin-embedded sections (A–H) or on cryostate sections (I–P, antibody pCFTR-C-1, 1:10,000 dilution) with a mainly intracytoplasmic staining pattern. In addition, especially, cryostate sections also had clear apical membrane staining, but cellular morphology and signal-to-noise ratio were clearly superior on formalin-fixed, paraffin-embedded tissues. Strong signals were found throughout the airways, including trachea (not shown) and large bronchi (A,B) and throughout the gastrointestinal tract from the cardiac glands of the glandular part of the stomach (C) and the duodenum (D) to all segments of the large intestine (E, colon; G, rectum). pCFTR protein was only weakly detected in the excretory ducts of the prostate gland (H). On cryostate sections, pCFTR was detected more apically, as seen in the nasal cavity (I) and the large bronchi (K). In addition, pCFTR was also found on cryostate sections within the excretory ducts of the parotid gland (M) and on the apical surface of some gall bladder epithelial cells (O). After preabsorption with the respective peptides used for immunization, signal intensities were abolished or markedly reduced (pCFTR-C-1, 1:15,000 dilution, S; pCFTR-N-1, 1:1500 dilution, W) when compared with non-absorbed antibodies (Q,U) or antibodies absorbed with an irrelevant peptide at the same concentration (R,V). Sections incubated with preimmune sera or four purified or non-purified immune sera from rabbits immunized with non-CFTR-related antigens at the same dilutions failed to develop any staining (F,J,L,N,P,T,X). Bars: E,F = 50 μm; Q–X = 25 μm; A,D,G–P = 10 μm; B,C = 5 μm.