Total RNA from mouse lungs was isolated using the Tri-Reagent protocol (Sigma, St. Louis, MO) and the yield and purity of RNA was determined electrophoretically and spectrophotometrically. After DNase treatment with RQ1 RNase-Free DNase (Promega, M160A), 2 mg of RNA were reverse transcribed into cDNA using M-MLV Reverse Transcriptase (Promega, M170B) and random primers (Invitrogen, 58875) according to the manufacturer's instructions. The primer pairs for real-time PCR were designed using Beacon Designer v7.01 software (Premier Biosoft International, Palo Alto, CA). Sequences for the primer pairs used are given in table S1. The PCR reactions contained SYBRH Green ER TM qPCR Super Mix Universal (Invitrogen, 11762-500), 200 nM of each primer (Invitrogen, Carlsbad), and 0.2 ml of cDNA template in a 20 ml reaction volume. RT-qPCR cycling parameters were initial incubation at 50uC for 2 minutes, denaturation at 95uC for 10 minutes followed by 50 cycles of 15 seconds at 95uC and 40 seconds at 60uC. Data collection was carried out using a Chromo4 Real-Time PCR detector (BioRad) and analyzed with Opticon Monitor 3 expression analysis software (BioRad). Relative levels of mRNA expression were calculated according to the DDCt method [34] .