Protein Expression Analysis of Lung Tissue

Triton X-100 lysis buffer (containing protease- and phosphatase-inhibitors) was used to lyse lung tissue or cells, as previously described (Catravas et al., 2010 (link)). Then, the samples were centrifuged at 20,000 g at 4°C for 20 min and the supernatant was used to calculate the protein concentration by the BCA Protein Assay (Thermo Fisher, Waltham, MA). Tissue and cell extracts (17 μg) were separated using 4–20% Tris-SDS-Glycine PAGE, transferred to Immuno-Blot PVDF membrane by electrophoresis (Bio-Rad Laboratories, Hercules, CA), and then blocked in a Tris-buffered saline solution containing 5% nonfat milk. The membranes were probed with antibodies against SOX18 mouse (Santa Cruz, Dallas, TX) and rabbit (Thermo Fisher, Waltham, MA), Claudin5 mouse (Thermo Fisher, Waltham, MA), pNF-κB pS536 rabbit (Cell Signaling, Danvers, MA), NF-κB rabbit (Cell Signaling, Danvers, MA) and the corresponding secondary antibodies against rabbit and mouse (Thermo Fisher, Waltham, MA). Protein expression was normalized by re-probing with anti-β-actin (Sigma Aldrich, Burlington, MA). Reactive bands were visualized using chemiluminescence (Super Signal West Femto; Pierce, Rockford, IL) on a LI-COR Odyssey image station (Lincoln, NE). Bands were quantified using LI-COR Image Station software.