placed into whole-body plethysmographs and interfaced with computers using differential pressure transducers. Mice were challenged for 3 min with a series of aerosolized methylcholine inhalations. Enhanced pause (Penh) was monitored for 3 min after each aerosol challenge by transducer (model TRD 5100, Buxco Electronics Inc., Wilmington, NC) connected to preamplifier modules (model MAX2270, Buxco Electronics Inc., Willington, NC). Penh is a function of total pulmonary airway during the respiratory cycle and is described by the following equation: Penh = pause x (PEP/PIP), Pause, PEP and PIP are expiration time, the peak expiratory pressure, and peak respiratory pressure, respectively. Lung and bronchoalveolar lavage samples Lung tissues and BAL samples were obtained as previously described [26]. Briefly, mice were anesthetized, the trachea was isolated by means of blunt dissection, and small-caliber tubing was inserted and secured in the airway. Two successive volumes of 1 ml of PBS were instilled and gently aspirated and pooled. The BAL fluids were immediately centrifuged at 6000 rpm at 4uC for 5 min, and supernatants were stored at 280uC until use. After removing the supernatant, the cells were counted manually in an Improved Neubauer (Hausser Scientific, Horsham, PA) chamber. Cytocentrifuged preparations (Cytospin 2, Cytospin, Shandon, UK) were stained with May-Gru¨ nwald-Giemsa for differential cell counts and examined under bright-field optical microscopy using a light microscope, and corresponding digital images were captured for subsequent analysis by a Spot CCD Camera driven by Advanced Spot RT Software version 3.3 (Diagnostic Instruments Inc., MI, USA). The lung was perfused with PBS through the right ventricle until the lung was clean. The lung was excised for RNA and protein analyses and/or inflated with fixative for histology. Western blot analysis Lung tissues were lysed with a lysis buffer (62.5 mM Tris-HCl pH 7.5; 500 mM NaF; 100 mM Na3VO4, and proteinase inhibitor cocktail, BD BioSciences, San Jose, CA) and then centrifugated at 10,0006g at 4uC for 30 min to obtain the cellular proteins in the supernatant. The protein concentrations were determined by BCA Protein Assay (Pierce Biotechnology Inc., Rockford, IL), and 20 mg of total protein from each sample were boiled for 5 min and electrophoresed on 4–20% Tris-Glycine gels (Invitrogen, Carlsbad, CA), transferred to nitrocellulose mem- branes, and blocked in blocking buffer (150 mM NaCl in 10 mM Tris, pH 7.5 containing 5% non-fat dry milk) for 1 hr at room temperature. The membranes were blotted with anti-EGFP (1:200; Santa Cruz Biotechnology, CA) or anti-HRF (in house prepared monoclonal antibody [27]) at 4uC for at least 16 hours, washed three times (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, and 0.1% Tween 20), incubated with HRP-conjugated goat anti- mouse IgG secondary antibody (1:5000 dilution, GE Healthcar- e,UK) for 1 h at room temperature, washed three times, followed by the detection of signal with SuperSignalH West Pico Chemiluminescent Substrate (Pierce Chemical Co., Rockford, IL, USA). The density of each protein band was scanned using the Bio-Rad Gel Doc system and the Quantity One 4.4.1 software (Bio-Rad Laboratories, Hercules, CA) and compared by densi- tometry to positive control. Immunofluorescence microscopy Freshly dissected lungs were fixed in Protocol Safefix II (Fisher Scientific Co., Kalamazoo, MI) and embedded in paraffin. The lung tissue sections (5 mm in thickness) were deparaffinized with xyline, rehydrated gradually with graded alcohol solution (100%, HRF Transgenic Mice Phenotype 95%, and 80%), and then washed with deionized water and immersed in 3% BSA for 1 h to block nonspecific binding. These slides were then incubated with primary mouse anti-HRF antibody at dilutions of 1:200 for 18 h at 4uC, washed twice in PBS/Tween-20 solution, incubated with a Texas Red-conjugated secondary antibody for 1 h at room temperature, and photo- graphed with a fluorescent microscope. We chose anti-HRF antibody to directly measure HRF. Therefore, an anti-GFP antibody was unnecessary. Histological examinations of lung tissues Lung tissues were fixed in Protocol Safefix II (Fisher Scientific Co.), and processed by AML Laboratories (Baltimore, MD). Briefly, samples were embedded with paraffin, sectioned at 5 mm, deparaffinized, dehydrated, and stained with hematoxylin and eosin (H&E). In collaboration with Dr. Allen Myers, Head of the Johns Hopkins Histologic Core, these specimens were examined under bright-field optical microscopy using a light microscope, and corresponding digital images were captured for subsequent analysis by a Spot CCD Camera driven by Advanced Spot RT Software version 3.3 (Diagnostic Instruments Inc.). Reverse transcription polymerase chain reaction (RT-PCR) Lungs were rapidly dissected and frozen on dry ice. For extraction of total RNA, tissues were homogenized in 1 ml of ice- cold TRIzol reagent (Invitrogen) and the total RNA was reverse- transcribed into cDNA using a random hexamer and a GeneAmp RNA PCR Kit (Applied Biosystems, Foster City, CA). PCR analysis was performed by AccuPower PCR PreMix kit (BIO- NEER, Alameda, CA) with each amplified primer set (EGFP-F, 59-GAC GTA AAC GGC CAC AAG TT-39; EGFP-R, 59-GAA CTC CAG CAG GAC CAT GT-39; actin-F, TCC TGT GGC ATC CAG GAA ACT; actin-R, GGA GGA ATG ATC CTG ATC TTC; IL-4-F, 59-TCA TCG GCA TTT TGA ACG AG-39; IL-4-R, 59-GAA TCC AGG CAT CGA AAA GC-39; IL-13-F, 59- TCA GCC ATG AAA TAA CTT ATT GTT TTG T-39; IL-13- R, 59-CCT TGA GTG TAA CAG GCC CAT TCT-39; MCP-1- F, 59-ACC AGC CAA CTC TCA CTG AAG C-39; MCP-1-R, 59-CAG AAT TGC TTG AGG TGG TTG TG-39; MDC-F, 59- CCT GGT GGC TCT CGT CCT TC-39; MDC-R, 59-CAG GGG ATG GAG GTG AGT AA-39) under formulated conditions. Amplified PCR products were analyzed by means of electrophoresis, and the intensity of the bands and the ratio of specific mRNA to b-actin were analyzed with the Bio-Rad Gel Doc system and the Quantity 4.4.1 software (Bio-Rad Laboratories). Quantification of total IgE, OVA-specific IgE, and chemokine levels Blood drawn from the heart and BAL fluids were used for measurement of total IgE levels with commercial mouse IgE isotype-specific enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (BD Biosciences, San Diego, CA). The levels of OVA-specific IgE in serum were determined using a commercial ELISA per the manufacturer’s instructions (MD Biosciences, Zu¨ rich Switzerland). IL-4 and eotaxin levels of BAL fluids were quantitated using commercial ELISA kits (R&D System, Inc, Minneapolis, MN) according to the manufacturer’s instructions. Statistical analysis All experiments were repeated with multiple mice and matched littermate controls as indicated by the n values in each experiment. PLoS ONE | www.plosone.org 3 June 2010 | Volume 5 | Issue 6 | e11077