P plcγ1

Serum starved PAE-KDR cells were incubated for 10 min with 1.5 nM VEGF (in all experiments VEGF-A₁₆₅ was used) with or without 30 min pretreatment with scFvs. Cells were lysed with lysis buffer (50 mM Tris (pH 7.5), 100 mM NaCl, 0.5% (w/v) Triton X-100) supplemented with protease inhibitor cocktail (Complete Mini EDTA-free, Roche) and phosphatase inhibitors (200 μM Na₃VO₄, 20 μM phenylarsine oxide). Lysates were diluted with 5× loading buffer (0.25 M Tris-HCl pH 6.8, 0.5 M DTT, 10% SDS, 50% glycerol, 0.5% bromophenol blue). Samples were boiled at 50 °C for 30 min, and resolved by 7% SDS PAGE, transferred to PVDF membranes (GE Healthcare, Piscataway, NJ, USA), and immunodecorated with primary antibodies (dilution 1:1000), followed by secondary alkaline phosphatase-coupled antibodies (1:10000). Immunoblots were developed with Novex AP Chemiluminescent Substrate (Invitrogen, Carlsbad, CA, USA). Amersham Imager 600 (GE) was used for analysis. Antibodies used were as follows: pVEGFR-2 (2478, Cell Signaling), tVEGFR-2 (2479, Cell Signaling), pPLCγ1 (2821, Cell Signaling), tPLCγ1 (2822, Cell Signaling), pAKT (4060, Cell Signaling), tAKT (4051, Cell Signaling), p38 (4631, Cell Signaling), and tp38 (9212, Cell Signaling). Protein marker used was PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa (26619, ThermoFisher, Waltham, MA, USA).

IMDM, penicillin, and streptomycin were purchased from Hyclone (Logan, UT, USA). Bovine serum albumin, PMA, U73122, PD98059, PF-02341066, and A23187 were purchased from Sigma (St. Louis, MO, USA). Anti-mouse IL-6 antibody and biotinylated anti-mouse IL-6 antibody were purchased from BD Biosciences (BD Pharmingen, San Diego, CA, USA). p-STAT3, STAT3, p-JAK, JAK, p-PLCγ1, PLCγ1, p-AKT, AKT, p-ALK, ALK, β-actin, p-ERK, ERK, p-JNK, JNK, p-p38, and p38 antibodies were all purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). TNF-α, CSF2, IL-6, IL-6R, IL-8, and GAPDH oligonucleotide primers were purchased from Bioneer Corp. (Daejeon, Korea).

Antibodies against the indicated proteins were purchased from Cell Signaling Technology: Lck (Cat. 2752), pSrc (Cat. 2101), pZAP70 (Y319, Cat. 2717), pZAP-70 (Y493, Cat. 2704), ZAP-70 (Cat. 3165), pErk1/2 (Cat. 9101), Erk1/2 (Cat. 4695), GAPDH (Cat. 5174), Calnexin (Cat. 2679), LAT (Cat. 9166), pLAT (Cat. 3581), PLC-γ1 (Cat. 2822), pPLCγ1 (Cat. 2821), SLP-76 (Cat. 70896) and GRB2 (Cat. 3972). Antibodies against pSLP-76 were purchased from Invitrogen, Cat. PA5-39759. The following antibodies were used for immunoprecipitation: anti-Lck clone 3A5 (EMD Millipore, Cat. 05-435) and anti-ZAP-70 clone 35A.2 (EMD Millipore, Cat. 05-253). The following reagents were used: Anti-human CD3 antibody (OKT3) (eBioscience, Cat. 14-0037-82), Pierce Protein A agarose beads (Thermo Scientific, Cat. 20333), Hydroxylamine hydrochloride (Sigma, Cat.159417), Methyl methanethiosulfonate (MMTS) (Sigma, Cat. 208795), n-Dodecyl β-D-maltoside (DDM) (Sigma, Cat. D4641), EZ-Link HPDP-biotin (Thermo Scientific, Cat. 21341), Streptavidin agarose beads (Invitrogen, S951), Poly-L-lysine (Sigma, Cat. P4707), Phosphatase Inhibitor Cocktail 2 (Sigma, Cat. P5726), Complete Protease Inhibitor Cocktail tablets (Roche, Cat. 11836170001) and ML211 (Cayman Chemical, Cat. 17630).

DOPAL (Purity: ≥90%) was acquired from Cayman Chemical (Ann Arbor, MI, USA). MOPAL (99%), MOPET (99%), IPy (≥97%), IPA (≥99.0%), ILA (99%), IBA (≥99.0%), dimethyl sulfoxide (DMSO), and bicinchoninic acid (BCA) were obtained from Sigma–Aldrich (St. Louis, MO, USA).
HUVECs, endothelial cell growth medium-2 (EGM-2) and endothelial basal medium (EBM) were obtained from Lonza (Slough, UK). Recombinant human VEGFA₁₆₅ was bought from R&D Systems (Minneapolis, MN, USA).
A PathScan^® Phospho-VEGFR-2 (Tyr1175) sandwich ELISA kit was purchased from Cell Signaling Technology (Hitchin, UK). NuPAGE lithium dodecyl sulfate (LDS) sample buffer and NuPAGE DTT and 4–12% Bis-Tris gels were obtained from Invitrogen (Loughborough, UK). Nitrocellulose 0.2 µm membranes was acquired from Bio-Rad (Hercules, CA, USA); p-PLCγ1, PLCγ1, p-Akt, Akt, p-eNOS, eNOS from Cell Signaling Technology (Danvers, MA, USA) and SuperSignal West Pico chemiluminescent substrate from Thermo Scientific (Hitchin, UK).

Standard samples (chlorogenic acid and γ-aminobutyric acid (GABA)) for high-performance liquid chromatography (HPLC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution, lipopolysaccharide (LPS), vitamin C, and dexamethasone were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Recombinant human interferon (IFN)-γ and tumor necrosis factor (TNF)-α were obtained from BioLegend (San Diego, CA, USA). Fetal bovine serum (FBS), Dulbecco’s modified Eagle medium (DMEM), and Roswell Park Memorial Institute (RPMI) 1640 medium were purchased from Gibco BRL (Rockville, MD, USA). Enzyme-linked immunosorbent assay (ELISA) kit for histamine was obtained from Abcam (Cambridge, UK) for histamine. Antibodies against p-p38, p38, p-ERK, ERK, p-JNK, JNK, p-Lyn, Lyn, p-Syk, Syk, p-Fyn, Fyn, p-PLCγ1, PLCγ1, p-PKCδ, PKCδ, NF-κB p65, Lamin B, Nrf2, HO-1, NQO1, iNOS, COX-2, and β-actin were purchased from Cell Signaling Technology (Danvers, MN, USA) or Santa Cruz (Dallas, TX, USA).