Cell line lysates were prepared in RIPA buffer as previously described 15 (link),16 (link). Protein pellets of surgically resected human squamous cell lung cancer specimens were obtained from the Lung Cancer Biospecimen Resource Network (LCBRN, University of Virginia). The dried protein precipitate pellet was suspended in buffer containing 5% SDS, 1% Triton, 50 mmol/L Tris, and 150 mmol/L sodium chloride containing a Complete Mini Protease Inhibitor Tablet (Roche, Indianapolis, IA) and sonicated for 7–10 sec on ice. Protein was quantified by the Pierce BCA protein assay kit (ThermoFisher Scientific). All protein samples were electrophoretically resolved through 4–20% gradient gels (Bio-Rad, Hercules, CA). Antibodies used were PIAS3 (#4164; Cell Signaling Technology, Danvers, MA), STAT3 (#sc-7179, Santa Cruz Technology, Santa Cruz, CA), and β-Actin (#A-5441, Sigma-Aldrich, St. Louis, MO). The working antibody dilutions were: PIAS3 (1:1000), STAT3 (1:2000), β-actin (1:50,000). Secondary antibodies were used at 1:10,000.
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