Irdye 680 labeled goat anti rabbit igg

Antibodies used were anti-phospho-Stat3 (Tyr705) (Cell signaling Technology, Cat # 9145), anti-E-cadherin (Cell signaling Technology, Cat # 3195), anti-Occludin (BD Transduction Laboratories, Cat # 611091), anti-Vimentin (Millipore, Cat #CS207806), anti-β-Actin (Sigma, Cat #A1978), anti-GAPDH (Santacruz Biotechnology, Cat # Sc-365062), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell signaling Technology, Cat #4370), anti-Axl (C89E7) (Cell signaling Technology, Cat #8661). Briefly, cells were rinsed in phosphate buffered saline (PBS) and lysed in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100 (v/v), 2 mM EDTA, pH 7.8 supplemented with 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 μg/ml aprotinin, and 10 μg/ml leupeptin). Protein concentrations were determined using the BCA protein assay (Pierce, Rockford, IL) and immunoblotting experiments were performed using standard procedures. For quantitative immunoblots, primary antibodies were detected with IRDye 680-labeled goat-anti-rabbit IgG or IRDye 800-labeled goat-anti-mouse IgG (LI-COR Biosciences, Lincoln, NE) at 1:5000 dilution. Bands were visualized and quantified using an Odyssey Infrared Imaging System (LI-COR Biosciences).