Anti phospho fak

The human OC cell lines CaOV3 and OVCAR3 were obtained from the American Type Culture Collection (Manassas, VA) and maintained in a humidified 5 % CO₂ incubator at 37 °C. Cells were passaged twice weekly. OVCAR3 cells were maintained in RPMI-1640 (Wisent, St-Bruno, QC, Canada) supplemented with 20 % FBS, insulin (10 mg/L), glutamine (2 mM) and antibiotics. CaOV3 cells were cultured in DMEM/F12 (Wisent) supplemented with 10 % FBS, 2 mM glutamine and antibiotics. Acellular ascites fractions were obtained at the time of initial cytoreductive surgery from women with advanced serous ovarian carcinomas. Samples were supplied by the Banque de tissus et de données cliniques et biologiques sur les cancers gynécologiques et du sein de Sherbrooke as part of the Banque de tissus et de données du Réseau de Recherche en Cancer des Fonds de Recherche du Québec en Santé (FRQS) affiliated to the Canadian Tumor Repository Network (CTRNet). HRP-conjugated anti-mouse and rabbit antibodies and anti-FAK antibody were purchased from Cell Signaling Technology (Danvers, MA). Anti-phospho FAK and anti-phospho Pyl2 were from Thermo Fisher (Waltham, MA). Anti-Pyk2 and anti-Tubulin were purchased from Sigma-Aldrich (Oakville, ON). CCL18 and CCL18 neutralizing antibody were from RnD Systems (Minneapolis, MN). Plasmid pCMV6-ENTRY-PTK2B was obtained from Origene (Rockville, MD).

Protein extracts from NSCLC cell lines and fresh frozen lung tissue samples were prepared in lysis buffer (0.5% Triton X-100, 50 mM β-glycerophosphate, pH 7.2, 0.1 mM dithiothreitol, 2 µg/mL leupeptin, and 2 µg/mL aprotinin). Extracts were resolved on 10% SDS-PAGE gels and transferred onto nitrocellulose membranes. Membranes were blocked in Tris-buffered saline (TTBS) containing 10 mM Tris-Cl (pH 7.4), 140 mM NaCl, 0.1% Tween 20, and 3% nonfat dry milk for 1 hour and incubated with TTBS containing indicated antibodies at 0.5 µg/mL for 12–16 hours at 4 ^oC. The following antibodies were used for immunoblotting: anti-KCNF1 (Sigma, HPA014738), anti-ITGB4 (Abcam, ab182120), anti-HA (Cell Signaling, 2367), anti-His (Proteintech, 10001-0-AP), anti-phospho-FAK (Tyr397, Invitrogen, 44624 G), anti-FAK (Invitrogen, AHO0502), anti-phospho-AKT (pS473, Cell Signaling, 9271), anti-AKT (Cell Signaling, 9272), anti-caspase 3, -caspase 7, and -caspase 9 (Cell Signaling: 9662, 9492, 9508), anti-GAPDH (Cell Signaling, 5174), and anti-Actin (Sigma, A2066). The membranes were extensively washed in TTBS and bound antibodies were visualized with horseradish peroxidase (HRP)-coupled secondary antibodies and ECL western blotting detection reagent (Amersham, RPN2106).

anti Fyn (sc-16), and anti phospho-paxillin (sc-101774; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Anti - and anti general-Akt (#9721 and #2938, respectively; Cell signaling Technology, Danvers, MA, USA). Anti FAK (#AHO0502; Biosource International (Camarillo, CA, USA). Anti phospho-FAK (#44625G; Invitrogen, Carlsbad, CA, USA). Anti actin (#MAB1501; Millipore, Temecula, CA, USA). Anti paxillin (#610052; BD Transduction Laboratories, San Diego, CA, USA). Rodhamine Phalloidin (Molecular Probes, Eugene, OR,USA)