Nbp2 29463

Lung tissue and cells were homogenized in lysis buffer (Cell signaling, USA). Samples were centrifuged at 14000g for 10 min at 4°C to remove debris. Protein concentration was determined using the DC protein assay from Bio-Rad. Samples for SDS-PAGE were denatured in a reducing Laemmli loading buffer at 99°C for 10 min. 25-30 µg of total protein per lane was electrophoretically separated in 12% TGX Stain-free Acrylamide gels and transferred into PVDF membranes (Pall Corporation, Germany) by a semidry-blotting method. The membranes were blocked using 6% non-fat milk in TBST buffer for 1 hour. Following, primary antibodies were used: anti-Shc (dilution 1:1000, 610879, BD Transduction Laboratories, USA), anti-p21 (dilution, 1:500, NBP2-29463, Novus Biologicals, USA), anti-LaminB1 (dilution 1:1000, #13435, Cell Signaling Technology, USA) and anti-β-actin (dilution 1:50000, A2228, Sigma-Aldrich, USA). Secondary antibodies were used: anti mouse (dilution 1:5000, W402B, Promega Madison, USA), and anti-rabbit (dilution 1:5000, W401B, Promega Madison, USA)

Paraffin sections with thicknesses of 3 µm were processed for immunohistochemistry. For labeling with primary antibodies, the sections were deparaffinized and treated for 30 min with 0.3% H₂O₂ in methanol. All the sections were heated in an autoclave for 20 min in a citrate buffer (pH 6.0), then incubated with the primary antibodies overnight. The primary antibodies and conditions are as follow: anti-p21 (1:25, mouse monoclonal, NBP2-29463, Novus Biologicals, CO, USA); anti-Ki-67 (1:800, mouse monoclonal, M7248, Dako, CA, USA), anti-CD31 (1:60, goat monoclonal, AF3628, R&D Systems, MN, USA), and anti-desmin (1:80, mouse monoclonal, M0760, Dako, CA, USA). Sections were stained with biotinylated anti-mouse IgG or anti-goat IgG (Dako, CA, USA) and visualized using H₂O₂-containing diaminobenzidine buffer.