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2 protocols using anti phospho vegfr2 tyr1175

1

Signaling Pathways Modulation Assay

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The TT cells were treated for 4 hrs in 60 mm cell culture dishes with the IC50 concentrations of the drugs, alone and in combination. Proteins were extracted and centrifuged, and the supernatant was collected. Total proteins were quantified, separated by SDS/PAGE, electro-blotted onto nitrocellulose membranes and saturated in 5% fat-free dried milk. Membranes were incubated overnight with primary antibodies and then incubated with secondary ones (Sigma-Aldrich). Immunoreactivity was detected with Euroclone ECL long-lasting substrate (Euroclone, Milano, Italy). The primary antibodies were anti-Erk1/2, anti-phospho-Erk1/2 (Thr202/Tyr204), anti-Akt, anti-phospho-Akt (Ser473), anti-VEGFR2, anti-phospho-VEGFR2 (Tyr1175), all (1:1000) from Cell Signaling (Danvers, MA, USA), and anti-β-actin (1:5000) from Sigma-Aldrich. Films were scanned and the band intensity was quantified with ImageJ software 1.44p. All experiments were performed in triplicate.
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2

Angiogenic Potential of Vestaine Compounds

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HuMedia-EG2 and VascuLife EnGS Comp Kit were purchased from KURABO and fetal bovine serum (FBS) from Hyclone. Medium-199 (M199), collagen-I solution, and Alamar blue were purchased from Thermo Fisher Scientific Inc. CELLBANKER-1 was purchased from Takara. Hexadimethrine bromide, antiβ-tubulin antibody, and Evans Blue were purchased from Sigma. Cignal™ Lenti Positive Control (GFP) and RNeasy mini kit were purchased from Qiagen. Growth factor reduced Matrigel was purchased from Corning. Recombinant human VEGF165 and recombinant human FGF-basic (bFGF) were purchased from Peprotech. PD98059 and VEGF receptor 2 (VEGFR2) inhibitor were purchased from Calbiochem. Complete EDTA-free was purchased from Roche and phosphatase inhibitor cocktail from Nacalai Tesque, Inc. LY294002, antiphospho-Akt (Ser473), anti-Akt, phospho-ERK1/2 (Thr202/Tyr204), anti-ERK1/2, anti-phospho-VEGFR2 (Tyr1175), and anti-VEGFR2 antibodies were purchased from Cell Signaling Technology, Inc. ECL prime Western Blotting Detection Reagents were purchased from GE Healthcare Life Sciences. Power SYBR Green PCR Master Mix and Taqman Gene Expression Master Mix were purchased from Applied Biosystems. Vestaine A 1 (natural-1) and vestaine B 1 were isolated from the extract of an actinomycete strain, Streptomyces sp. SANK 63697. All other chemicals used were of reagent grade.
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