Anti phospho src

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).

Preparation of cell lysates and immunoblot analysis was performed via established protocols [82 (link)]. Anti-human primary antibodies were implemented at a 1:1, 000–2, 000 dilution and include: anti-NDRG1 (Cat#:ab37897) from Abcam (Cambridge); anti-Src (Cat#:2123), anti-phospho-Src Family (Tyr416; Cat.#:6943), anti-phospho-Src(Tyr527; Cat.#:2105), anti-p130Cas (Cat.#:13846), anti-phospho-p130Cas (Tyr249; Cat.#:4014), anti-phospho-p130Cas (Tyr410; Cat.#:4011), anti-EGF Receptor (Cat.#:2926), anti-phospho-EGF Receptor (Tyr1148; Cat.#:4404), anti-c-Abl (Cat.#:2862), anti-phospho-c-Abl (Tyr245; Cat.#:2861), anti-PAK1 (Cat.#:2602), anti-phospho-PAK1 (Thr423; Cat.#:2606), anti-PTP-PEST (Cat.#:4864), anti-PTP1B (Cat.#:5311), anti-CrkII (Cat.#:3492) and anti-phospho-CrkII (Tyr221; Cat.#:3491) were from Cell Signaling Technology (Boston, MA); and anti-Rac1 (Cat.#:05–389) was from Millipore (Darmstadt). The secondary antibodies (1:10, 000 dilution) included: anti-goat (Cat.#:A5420), anti-rabbit (Cat.#:A6154) and anti-mouse (Cat.#:A4416) antibodies from Sigma-Aldrich. β-actin (1:10, 000; Cat.#:A1978, Sigma-Aldrich) was used as a loading control.

Cytoplasmic cell fraction was collected by using cell lysis buffer (20 mM Tris-HCl (pH 8.0; Wako), 2 mM EDTA (Wako), 0.5% NP-40 (Wako), 1 μM pepstatin (Sigma), 1 μM leupeptin (Sigma), 2 mM sodium orthovanadate (Wako), 1 μM calpain inhibitor (Sigma), phosphatase inhibitor cocktail I/II (Sigma), and 1 mM phenylmethylsulfonyl fluoride (Sigma)). The protein contained amount of these fraction was evaluated using a bicinchoninic acid protein-assay kit (Wako). The extracts (40 μg of protein) were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinyl difluoride membranes (GE Healthcare, Buckinghamshire, UK). The membranes were reacted with the following antibodies: anti-Bcl-2, anti-Bcl-xL, anti-Survivin, anti-MDR1, anti-BCRP, anti-MRP1, anti-LRP1 (Santa Cruz Biotechnologies, CA, USA), anti-phospho-Src (Tyr527), anti-Src (Cell Signaling Technology, Beverly, MA), and anti-β-actin (Sigma) as an internal control. The membranes were reacted with horseradish peroxidase-coupled secondary antibodies (GE Healthcare) for 1 h at room temperature and proteins were assessed using a Luminata Forte (Merck Millipore, Nottingham, UK).

Proteins were extracted using RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS and 50 mM Tris pH. 7,5) with 1 mM PMSF, 10 mM NaF, 2 mM NaVO₃ and Protease Inhibitor cocktail (cat.no. 04-693-1160-001, Roche, USA). For analysis of phosphorylated histones, extracts were prepared in boiling 2% SDS buffer (in 150 mM Tris pH 6,8). Western Blot (WB) analysis was performed as previously described in [17 (link)]. The following antibodies were used: mouse monoclonal antibodies (Mab) to p140Cap already characterized in [17 (link)] (1:500), anti phospho-p130Cas (Tyr410; #4011, 1:1000), anti phospho-Src (Tyr416; #2101, 1:1000), anti phospho-JAK2 (Tyr1007/1008; #3776S, 1:1000), anti γH2AX (Ser139; #2577, 1:1000) and anti H2AX (#2595, 1:1000) from Cell Signaling, Beverly, MA; anti GAPDH (MAB374, 1:8000) from Millipore, Billerica, MA, USA; anti p130Cas (cat.no 610272, 1:2500) from BD Transduction Laboratories, Franklin Lakes, NY; anti Src (B-12, 1:1000) and anti JAK2 (sc-278, 1:1000) from Santa Cruz Biotechnologies, Palo Alto, CA, USA; anti Tubulin (T5168, 1:8000) from Sigma-Aldrich Co, Italy. Secondary antibodies conjugated with peroxidase and nitrocellulose membranes were purchased from GE Healthcare (Buckinghamshire, UK). When appropriate, the membranes were stripped according to manufacturers’ recommendations and re-probed.

Full-length wild-type Duoxa1 was amplified using PCR from mouse cDNA and ligated into the pMX-IRES-EGFP vector as pMX-Duoxa1 using the BamHI and XhoI (Enzynomics, Daejeon, Korea) sites. The following primers were used: Duoxa1-For, 5′-GCTAGGATCCATGGCTGCTCTTGGACACAC-3′ and Duoxa1-Rev, 5′-CGACTCGAGCAGGGAACAGTCGGACTCTTTG-3′. TRIzol reagent was obtained from Life Technologies (Carlsbad, CA, USA). A monoclonal β-actin (A5441) antibody and DAPI (D9542) were obtained from Sigma (St. Louis, MO, USA). Antibodies for anti-phospho-ERK-1/2, anti-total ERK-1/2, anti-phospho-p38, anti-total p38, anti-phospho-JNK, anti-total JNK, anti-phospho-IκB, anti-phospho-Akt, anti-Akt, anti-phospho-Src, anti-Src, and anti-Btk were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). Anti-Phospho-Btk (GTX61791) antibody was obtained from GeneTex (Irvine, CA, USA). Anti-c-Fos, anti-NFATc1, anti-IκB, and anti-PLCγ2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Duoxa1 antibody was obtained from Bioss Inc (BS-11433^®, Wobun, MA, USA). Donkey anti-rabbit and anti-mouse immunoglobulin secondary antibodies were purchased from Enzo Life Sciences (Farmingdale, NY, USA).