Western blot analysis of lung tissue

Frozen lungs were lysed in 1 ml of lysis buffer containing a protease inhibitor cocktail using an ULTRA-THURRAX. Lysates were cleared by centrifugation before total protein determination using the BCA protein assay. SDS–polyacrylamide gel electrophoresis and Western blotting were performed following standard procedures. Membranes were blocked and then probed with antibodies directed against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pAkt, and total Akt. The membranes were then washed in tris-buffered saline/Tween 20 followed by incubation with horseradish peroxidase (HRP)–conjugated secondary antibodies. Immunoreactive bands were detected by addition of an enhanced chemiluminescence substrate.