Anti β actin peroxidase conjugated antibody

Whole-cell protein extracts were prepared as previously described [20] . Cells were collected in a lysis buffer with protease inhibitors (20 mM Tris-HCl, 137 mM NaCl, 25 mM beta-glycerophosphate, 2 mM NaPPi, 2 mM EDTA, 1 mM Na 3 VO 4 , 1% Triton-X, 10% glycerol, 0.5 mM DTT, 1 mM PMSF, 15 µg/ml aprotinin and leupeptin, 2 mM benzamidine). Protein samples were resolved by SDS-PAGE, and transferred to hybond ECL nitrocellulose membranes (GE Healthcare Amersham, Buckinghamshire, UK). Antibodies recognizing phospho-STAT3 (Tyr705), total STAT3, and cleaved PARP were obtained from Cell Signaling (Beverly, MA, USA). Anti-cyclinD1 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and anti-β-actin-peroxidase-conjugated antibody was from Sigma-Aldrich (Munich, Germany). Anti-SERPINA3 antibody (LS-B5826) was from LIFESPAN Biosciences. Membranes were incubated with primary antibodies diluted in TBS-T (10 mM Tris/HCl, pH 8.0, 0.12 M NaCl, 0.1% Tween-20, and 0.05% sodium azide) containing 5% skimmed milk. Antibody recognition was detected with the respective secondary antibody either anti-mouse IgG, antirabbit IgG linked to horseradish peroxidase (Cell Signaling Technology, Beverly, MA, USA). Immunocomplexes were visualized using the enhanced chemiluminescence detection system (Thermo Scientific, Rockford, USA).