Anti phospho fak

Recombinant human TNF-α was purchased from R&D Systems (Wiesbaden-Norderstedt, Germany). Anti-PAR-1 (ATAP2), anti-NQO1, anti-eNOS, and anti-VCAM-1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-phospho-HDAC5, anti-HDAC5, anti-phospho-ERK5, anti-ERK5, anti-phospho-AKT, anti-AKT, anti-phospho-AMPK, anti-AMPK, anti-phospho-eNOS, anti-EEA1, anti-phospho-Src, anti-phospho-FAK, anti-phospho-Erk1/2, anti-Erk1/2 and anti-VE-cadherin were from Cell Signaling Technology (Danvers, MA, USA). Anti-COX-2 was from Cayman Chemical Company (Ann Arvor, MI, USA). Anti-tubulin was from Sigma–Aldrich (St. Louis, MO, USA). Phenylephrine, Acetylcholine and U46619 were purchased from Sigma–Aldrich (St. Louis, MO, USA). Pertussis toxin (PTX) was purchased from Gibco. Small interfering RNA against human PAR-1 was purchased from Santa Cruz Biotechnology (sc-36663, Santa Cruz, CA, USA). For PAR-1 silencing, HUVECs were transiently transfected with 100 pmol/L of control RNA or siRNA targeting human PAR-1 using Lipofectamine 2000 reagent (#11668-019, Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. A non-specific control siRNA from Bioneer was used as a negative control. HUVECs were harvested 48–72 hours after siRNA transfection, protein expressions were assessed by immunoblotting with antibodies and mRNA levels by quantitative real time RT-PCR (RT-qPCR).

Analysis of proteins was performed by Western blot as previously described [25 (link)]. Briefly, 30 μg protein of whole cell lysates were subjected to SDS-PAGE electrophoresis in 4 to 20% polyacrylamide gels (Bio-Rad, Hercules, CA, USA). Proteins were transferred onto nitrocellulose membranes (Bio-Rad), incubated overnight at 4 °C with primary antibodies (1:1000), followed by washes and incubation with the appropriate secondary antibodies for 1 h (1:2000). Protein bands were developed in autoradiography films (Denville Scientific Inc., South Plainfield, NJ). Antibodies used in this study were purchased from Cell Signaling Technology and included anti-EGF Receptor (D38B1), anti-HER3/ErbB3 (D22C5), anti-phospho-EGFR (Tyr1068; D7A5), anti-phospho-HER3/ErbB3 (Tyr1289; D1B5), anti-phospho-FAK (Tyr925), anti-phospho-Stat3 (Tyr705), anti-Stat3 (79D7), anti-phospho-AKT (S4737), and anti-AKT (C67E7).

Autophagy inducer PP242 was purchased from Selleck chemicals (Houston, TX). Anti-CXCR4-APC (eBioscience, San Diego, CA, anti-VLA-4-APC (BD Bioscience, San Jose, CA) and anti-VCAM-1-APC (Biolegend, San Diego, CA) were used for FACs analysis. The following antibodies were used for Western blot: anti-Gli1 (Cell Signaling, Danvers, MA), anti-Gli2 (Santa Cruz, Dallas, TX), anti-Ptch1 (Santa Cruz, Dallas, TX), anti-FAK (AbCam, Cambridge, MA), anti-phospho-FAK (Cell Signaling, Danvers, MA), anti-paxillin (BD Biosciences, San Jose, CA), anti-phospho-paxillin (Cell Signaling, Danvers, MA), anti-SDF-1 (Santa Cruz, Dallas, TX), anti-IL-6 (R&D Systems, Minneapolis, MN), anti-LC3 (Novus Biologicals, Littleton, CO), and anti-GAPDH (Cell Signaling, Danvers, MA). Recombinant human SDF-1 (rhSDF-1) was purchased from Pepro Tech (Rocky Hill, NJ), and recombinant human IL-6 (rhIL-6) was purchased from R&D System (Minneapolis, MN). CXCR4 antagonist AMD3100, 2,7-dichlorodihydrofluorescein-diacetate (DCH-FDA, 50MG), ROS inhibitor N-acetyl-L-cysteine (NAC), FITC-labeled phalloidin, l-alpha-lysophosphatidylcholine, and autophagy inhibitor 3-MA were all purchased from Sigma-Aldrich (St Louis, MO).