determined using the cycle threshold method (StepOnePlus, Applied Biosystems). Muscle Ring Finger 1 (MuRF-1) and atrogin-1 expression were quantified according to the manufacturer (TaqMan). RNase protection assay 32P-labelled riboprobes were synthesized using commercial mouse multiprobe kits (BD Biosciences), protected RNA fragments were separated using a 5% polyacrylamide gel, and detected by autoradiography. Bands of individual mRNAs were quantified and normalized to the L32 housekeeping gene (FluorChem 8000, Alpha Innotech). ELISA and western blot assays An immunoassay kit was used to quantify NF-kB (p65 subunit) within nuclear extracts, according to the manufacturer’s instruc- tions (Trans-AM, Active Motif). To inhibit the NF-kB pathway, myotubes were treated with the IKK-2 inhibitor SC-514 (Calbiochem) at a dose of 100 mM [52], applied one hour prior to cytomix/LPS stimulation. Western blotting was used to evaluate activation of ERK1/2 MAPK, using antibodies against the total and phosphorylated forms of the protein (Cell Signaling). CFTR protein expression was evaluated in lysates from human skeletal muscle tissue, E6/E7 human muscle cells, Calu3 cells, and HEK cells with stable overexpression of CFTR. CFTR was immunoprecipitated with anti-CFTR antibody (clone M3A7) and anti-mouse Ig beads. The precipitates were separated using an 8% polyacrylamide gel, and then analyzed by western blotting with a second anti-CFTR antibody (clone 23C5-2) and the TrueBlot detection system (eBioscience). Pseudomonas aeruginosa lung infection Mice were infected by intratracheal inoculation at 16105 cfu using a mucoid strain encapsulated within agar beads, and euthanized at day 2 post-infection, as we have previously described in detail [53]. 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PLoS Genetics | www.plosgenetics.org 13 July 2009 | Volume 5 | Issue 7 | e1000586