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    To screen the proteins complicated in mouse lung ischemia, the lungs from B6 wild type mice were procured and subject to cold ischemia for 24 hours. The lung tissue lysates were made by adding lysis buffer [1% SDS, 10 mmol/L Tris-Cl (pH 7.6), 20 µg/µL aprotinin, 20 µg/µL leupeptin, and 1 mmol/L 4-(2-aminoethyl)benzenosulfonyl fluoride] after homogenizing with mortar and pestle in liquid nitrogen. The supernatants were obtainded after centrifugation at 12000 g for 15 minutes at 4. The protein concentrations were determined using the Bicinchoninic Acid Protein Assay kit (Pierce, Rockford, IL). Fiften micrograms of protein were separated on SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. After blocking, the membranes were incubated with the appropriately diluted primary antibody at 4°C overnight. After washing with TBST, the membranes were incubated with appropriately diluted HRP-conjugated secondary antibodies at room temperature for 1 hour. Proteins were detected with the enhanced chemiluminescence kit (Amersham Pharmacia Biotechnology, Inc., Piscataway, NJ).

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