Anti phospho 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).

Tris, NaCl, and SDS for molecular biology and buffer preparation were purchased from Sigma‐Aldrich. Cell culture media and supplements were from Invitrogen (La Jolla, CA, USA). Antibodies were purchased from commercial sources as follows: anti‐EGFR (IHC‐00005; Bethyl Laboratories, Montigny, TX, USA), anti‐phospho‐EGFR [Tyr1068, Tyr992, and Tyr845 (Cell Signaling, Danvers, MA, USA)], anti‐TRAF4 (sc‐1920; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti‐HA (H9658; Sigma‐Aldrich), Rabbit anti‐HA (Cell Signaling), mouse anti‐Omni and rabbit anti‐Omni (Santa Cruz Biotechnology), rabbit anti‐myc tag (Cell Signaling), mouse anti‐V5 antibody (Invitrogen), anti‐phospho‐ERK5 (Cell Signaling), anti‐ERK5 (Cell Signaling), anti‐phospho‐ERK1,2 (Santa Cruz Biotechnology), anti‐ERK1,2 (Cell Signaling), anti‐AKT (Cell Signaling), anti‐phospho‐AKT (Ser473; Cell Signaling), anti‐β‐actin (Cell Signaling). Recombinant human EGF was purchased from R&D (Minneapolis, MN, USA). Doxycycline and DMSO were purchased from Sigma‐Aldrich.