Anti erk5

Equal amounts of cell lysates were separated by 12 % SDS-PAGE, and electrophoretically transferred to PVDF membrane. The membrane was blocked and probed with primary antibody (anti-ASK1, anti-phospho-ASK1, anti-p38, anti-phospho-p38, anti-ERK1/2, anti-phospho-ERK1/2, anti-ERK5, anti-phospho- ERK5, anti-JNK, anti-phospho-JNK from Cell Signaling Technology; anti-MEKK2, anti-NIS, anti-TSHR from Santa Cruz; anti-β-actin from Sigma) followed by HRP (horseradish peroxidase)-labeled goat anti-mouse IgG (Abcam) or HRP-labeled goat anti-rabbit IgG (Abcam). Chemiluminescence was used to analyze protein levels and β-actin was used as a protein loading control. Semi-quantitative analysis was conducted by using ImageJ 1.49v (National Institutes of Health, USA).

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).

Whole-cell extracts from H460 cells were prepared using cell lysis buffer containing 25 mM HEPES pH 7.5, 0.3 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, 20 mM β-glycerophosphate, 0.1 mM Na₃VO₄, 0.1% Triton X-100 and complete protease inhibitor cocktail (P8340 Sigma, Madrid, Spain). Lysates were centrifuged and the supernatants transferred to a new tube. After adding SDS loading buffer the lysate was boiled for 5 min. 20 µg of protein extract was separated on SDS-polyacrylamide gel and electroblotted to Immobilon P membranes (Millipore, Burlington, VT, USA). Antibodies used were anti-p-ERK5 (3371, Cell Signaling, Danvers, MA, USA), anti-ERK5 (3372, Cell Signaling), anti-p-ERK1/2 (9106, Cell Signaling), anti-fibronectin (sc-71113, Santa Cruz Biotechnology, Dallas, TX, USA), anti-p120 (P17920, BD Biosciences, Franklin Lakes, NJ, USA), γ-catenin (610254, BD Transduction, Franklin Lakes, NJ, USA), anti-β-tubulin (T9026, Sigma, St. Louis, MO, USA), phospho-Smad2/3 (Ser 423/425) and Smad2/3 (FL-425) were from Santa Cruz Biotechnology (Dallas, TX, USA). The signal was detected using enhanced chemiluminiscent (ECL) method (Santa Cruz Biotechnology). After washes the blots were re-incubated with the α-tubulin antibody to normalize for protein load.