software identified this amino acid as an exposed or buried residue respectively with a non-structural role. Conservation could not be determined due to insufficient data. The score assigned by Conseq was validated using PolyPhen (http://www.bork.embl-heidelberg. de/polyPhen/). Cloning of hCAREx8 and site directed mutagenesis hCAREx8 was cloned from RNA isolated from primary human airway epithelia (Qiagen, Valencia, CA; hCAREx8F: 59GCGAA- TTCGCCACCATGGCGCTCCTGCTCTGCTTCG andhC- AREx8R: 59GTGGATCCTTATACAACTGTAATTCCATC). The fragment was digested with EcoRI and BamHI and cloned into pcDNA3.1(-) or an Ad5-CMV shuttle plasmid. DNA sequencing and Western blot confirmed the expected protein. hCAREx8 was modified with two FLAG tags (DYKDDDDK) added between amino acid 22 and 23, similar to previously described modifications in hCAREx7. Site- directed mutagenesis was performed to generate hCAREx7/8 according to manufacturer’s standard protocol (Stratagene, Cedar Creek, TX) with the following primer: GSIV-ITVV, 59-CCAGCACAGAG- CAAGGATATCACTGTAGTATAGGGATCCGAGCTC. Site-directed mutagenesis was performed to generate CAREx8/7 according to manufacturer’s standard protocol (Stratagene, Cedar Creek, TX) with the following primer: ITVV-GSIV, 59-CCTTA- CAAGACTGATGGAGGTTCAATTGTATAAGGATCAAG- GGTG Site-directed mutagenesis was performed to generate hCAREx8-PDZ according to manufacturer’s standard protocol (Stratagene, Cedar Creek, TX) with the following primer: ITVV-*TVV, 59-CCTTA- CAAGACTGATGGATAAACAGTTGTATAAGGATCAAG- GGTGG. Human airway epithelia Primary human airway epithelia were isolated from trachea and bronchi of donor lungs and seeded onto collagen coated, semi- permeable membranes (Millipore, Bedford, MA) and grown at the air-liquid interface as previously described [44,45]. Approximately two weeks after seeding, cultures were well-differentiated and attained a measurable transepithelial resistance. To augment endogenous hCAR expression, epithelia were transduced with adenovirus carrying hCAREx7 or hCAREx8 from the basolateral surface as previously described [23,41]. Amaxa transfection Primary airway epithelial cells seeded on plastic were trypsin- ized, washed and cells electroporated with 2.5 mg of plasmids encoding hCAREx7, hCAREx8 or eGFP using the Amaxa Nucleofector I (Amaxa Inc, Walkersville, MD) according to manufacturer’s standard protocol for primary mammalian epithe- lial cells (VPI-1005, program T-20). Approximately 36105 cells were seeded onto collagen coated, semi-permeable membranes as described above. Epithelia were infected with Ad-b-gal (MOI 10 pfu/cell) for 1 hr at 37uC, washed twice and lysed 48 hr post infection. b-galactosidase expression per mg protein was deter- mined as previously described [42]. Adenovirus infection Chinese hamster ovary cells were plated in 24 well dishes and transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. Twenty-four hours after transfection cells were infected with Ad serotype 5 containing the b-galactosidase gene (Ad-bGal) (MOI 100) for 1 hr at 37uC. 48 hours later cells were lysed and b-galactosidase expression per mg protein was determined as previously described [42]. Novel CAR Exon 8 Interactions Co-transfection in COS-7 cells COS-7 cells were electroporated by standard methodologies. Briefly, 10 million cells were mixed with 20 mg of plasmid DNA for single transfection, 15 mg of each DNA for double transfections, or 10 mg of each for triple transfections, in 400 ml of cytomix (120mM KCl, 0.15mM CaCl2, 10mM K2HPO4, 10mM KH2PO4, 25mM HEPES, 2mM EGTA, 5mM MgCl2, 2mM ATP and glutathione) and put in an electroporation cuvette (Bio-Rad Laboratories, Hercules, CA) for 30 minutes on ice. After electroporation, cells were seeded onto 10cm dishes for immunoprecipitation (IP) and collagen coated glass chamber slides for immunofluorescence studies 2 days later. Immunostaining COS-7 cells grown on collagen coated chamber slides or airway epithelia were washed once with PBS, fixed with 4% paraformal- dehyde, permeabilized with 0.1% Triton X-100, and blocked with 2% BSA in SuperBlock (Pierce, Rockford, IL). Cells were incubated with primary Ab, washed extensively and incubated with goat anti-mouse Alexa-568 secondary Ab. After washing, slides were coverslipped with Vectashield mounting media (Vector Laboratories, Inc, Burlingame, CA). Images were acquired with a BioRad MRC-1024 Laser Scanning Confocal Microscope (Her- cules, CA) mounted on a Nikon E600 microscope (Melville, NY) using a 60X oil immersion lens. Fluorescence imaging was performed on an Olympus IX71 X-Cite 120 fluorescence microscope (Center Valley, PA) followed by quantitation using Image J. Quantitative real time PCR RNA was isolated from primary airway cultures greater than 2 weeks old or primary lung tissue using TRIzol with the Pure Link RNA kit (Invitrogen). cDNA was synthesized using RT2 EZ First Strand Kit (SA Biosciences). Primers and probes for the seventh or eighth exon of CXADR were designed with Primer Express software (Applied Biosystems). The sequences for hCAREx7 were: hCAREx7-F, 59-TGCCAGAAGCTACATCGGCAGTAA; hCA- REx7-R, 59-ATAGACCCATCCTTGCTCTGTGCT; hCAREx7 probe, 59-AAGTCGAATGGGTGCGATTCCTGTGA (59 FAM, 39 TAMRA-SP); PCR product 141bp. The sequences for the hCAREx8 were: hCAREx8-F, 59-AGGGAAGATGTGCCACCTC- CAAA; hCAREx8-R, 59-CAACTGTAATTCCATCAGTCTTG- TAAG; hCAREx8 probe, 59-ACTGCCAGAAGCTACATCGG- CAGTAA (59 FAM, 39 TAMRA-SP); 165bp. Samples were run using TaqMan Fast Universal PCR Master Mix (Applied Biosystems) on a 7500 Fast Real-Time PCR System (Applied Biosystems). Transcript number was quantitated by plasmid standard curve. Abundance relative to 18s (Invitrogen, 115HM-02) or hGAPDH (Invitrogen, 100H-02) as the reference gene confirmed the presence of significantly more hCAREx7 transcripts than hCAREx8. Immunoprecipitation and Western blot Cells from two 100mm plates were placed on ice, washed once with ice cold PBS, and lysed with lysis buffer (50mM Tris-HCl, pH 7.5, 137mM NaCl, 1% Triton X-100, 5mM EDTA, 1mM EGTA, protease inhibitors (10 mg/ml) leupeptin, aprotinin, pepstatin, and 1mM phenylmethylsulfonyl fluoride) by rocking at 4uC. Cells were scraped, sonicated 5 times and spun in a microcentrifuge at full speed for 10 minutes. For co-immunopre- cipitation, supernatant was incubated with the indicated Ab with rotation at 4uC overnight. Protein A or G conjugated sepharose (Amersham Biosciences, Uppsla Sweden) was added for 1-2 hours followed by a wash with lysis buffer, 10% lysis buffer in TBS PLoS ONE | www.plosone.org 11 March 2010 | Volume 5 | Issue 3 | e9909