The following were used: Telmisartan (Tokyo Chemical Industry Co. Tokyo, Japan), Trypan Blue (Sigma-Aldrich, St. Louis, MO, USA), RPMI-1640 (Gibco-Invitrogen, Carlsbad, CA, USA), Fetal Bovine serum (FBS, Wako Pure Chemical Industries, Osaka, Japan), penicillin-streptomycin (Invitrogen, Tokyo, Japan), Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan), Cell Cycle Phase Determination kit (Cayman Chemical, Ann Arbor, MI, USA), Annexin V-FITC Early Apoptosis Detection kit (Cell Signaling Technology, Boston, MA, USA), protease inhibitor cocktail (Pro-Prep, complete protease inhibitor mixture; iNtRON Biotechnology, Sungnam, Korea), M30 Apoptosense ELISA kit (PEVIVA AB, Bromma, Sweden), Human Phospho-RTK Array kits and Angiogenesis Antibody Array kits (R&D Systems, Minneapolis, MN, USA). Telmisartan was prepared as a 10 mM stock solution in dimethyl sulfoxide (DMSO). The stock solutions were stored at −20°C.
Human phospho rtk array kit
Manufactured by R&D Systems
Sourced in United States, United Kingdom
The Human Phospho-RTK Array Kit is a semi-quantitative tool that allows for the simultaneous detection and comparison of the phosphorylation levels of multiple receptor tyrosine kinases (RTKs) in human samples. This array-based assay provides a broad overview of RTK activation status within a single experiment.
Automatically generated - may contain errors
Lab products found in correlation
Human phospho kinase array kit, by R&D Systems (5 mentions)
Human phospho mapk array kit, by R&D Systems (4 mentions)
Chemiluminescence detection system, by PerkinElmer (3 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (3 mentions)
P erk, by Cell Signaling Technology (3 mentions)
P akt, by Cell Signaling Technology (3 mentions)
Plx4720, by Selleck Chemicals (3 mentions)
Alamarblue cell viability reagent, by Thermo Fisher Scientific (2 mentions)
α tubulin, by Cell Signaling Technology (2 mentions)
Celltiter glo cell viability assay, by Promega (2 mentions)
Doxycycline, by Takara Bio (2 mentions)
Epidermal growth factor (egf), by R&D Systems (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Hb egf, by R&D Systems (2 mentions)
P rtk, by R&D Systems (2 mentions)
Sirna against mitf and non targeting control sirna, by Horizon Discovery (2 mentions)
Azd6244, by Selleck Chemicals (2 mentions)
U0126, by Selleck Chemicals (2 mentions)
Vemurafenib, by Selleck Chemicals (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
73 protocols using human phospho rtk array kit
1
Telmisartan's Anti-Cancer Effects
2
Phospho-RTK Array Analysis of Cell Lines
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Human p-RTKs were assayed using Human Phospho-RTK Array Kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Briefly, p-RTK array membranes were blocked with 5% BSA/TBS (0.01 M Tris-HCl, pH 7.6) for 1 h and incubated with 2 mL of lysate, which was prepared from cell lines after normalization to ensure equal protein amounts. The membranes were washed 3 times with TBS plus 0.1% v/v Tween-20 for 10 min each and 2 times with TBS alone for 10 min each to remove unbound materials. Then, the membranes were incubated with an HRP-conjugated anti-phospho-tyrosine antibody for 2 h at room temperature. The unbound HRP-conjugated antibody was washed away with TBS plus 0.1% Tween-20. Finally, each array membrane was exposed to X-ray film using a chemiluminescence detection system (PerkinElmer Co.). The immunoreactive bands were analyzed by densitometric scanning (TIc scanner, Shimizu Co., Ltd., Kyoto, Japan).
3
Profiling Kinase Phosphorylation Dynamics
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Phosphorylation of RTKs and other kinases was screened with protein array method using human phospho-RTK array kits and human phospho-kinase array kits (R&D Systems). Relative levels in cell lysates (300 μg per sample) were analyzed according to manufacturer's protocol and quantified with AlphaVIEW SA analysis software.
4
Phospho-RTK Array Analysis Protocol
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Human phosphorylated receptor tyrosine kinases (p-RTKs) were assayed using Human phospho-RTK Array Kits (R&D Systems), according to the manufacturer's instructions. Briefly, p-RTK array membranes were blocked with 5% bovine serum albumin/0.01 M Tris-HCl, pH 7.6 (TBS) for 1 h and then incubated with 2 ml of the lysate prepared from cell lines after normalization, so that the amounts of protein were equal. After three washes for 10 min each with TBS plus 0.1% v/v Tween-20 and two washes for 10 min with TBS alone to remove unbound material, the membranes were incubated with anti-phospho-tyrosine-horseradish peroxidase antibody for 2 h at room temperature. The unbound HRP antibody was washed out with TBS plus 0.1% Tween-20. Finally, each array membrane was exposed to an X-ray film using a chemiluminescence detection system (Perkin-Elmer Co.).
5
Profiling Phospho-RTK Landscape
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Human phospho-RTK was assayed using Human Phospho-RTK Array kits (R&D Systems, Minneapolis, MN, USA), according to the manufacturer's instructions. Briefly, p-RTK array membranes were blocked with 5% BSA/TBS (0.01 M Tris-HCl, pH 7.6) for 1 h and incubated with 2 ml of lysate, which was prepared from cell lines after normalization to ensure equal protein amounts. After 3 washes for 10 min each with TBS plus 0.1% v/v Tween-20 and 2 washes for 10 min with TBS alone to remove unbound materials, the membranes were incubated with anti-phospho-tyrosine-HRP antibody for 2 h at room temperature. The unbound HRP antibody was washed out with TBS plus 0.1% Tween-20. Finally, each array membrane was exposed to X-ray film using a chemiluminescence detection system (Perkin-Elmer Co.). The immunoreactive band density obtained via this array was analyzed by densitometric scanning (TIc scanner; Shimizu Co., Ltd., Kyoto, Japan).
6
Phospho-RTK Profiling for Cell Signaling
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The relative phosphorylation levels of 49 RTKs and two related proteins were measured using a Human Phospho-RTK Array Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions, with slight modifications. Briefly, cells were cultured in RPMI 1640 containing 10% FBS and lysed in array buffer prior to reaching confluence. The arrays were blocked with blocking buffer and incubated overnight with 300 μg of cell lysate at 4 °C before being washed, incubated with horseradish peroxidase (HRP)-conjugated phospho-kinase antibodies, and treated with SuperSignal West Dura Extended Duration Substrate (Pierce Biotechnology, Rockford, IL, USA).
7
Phospho-RTK Array Kit Protocol
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A Human Phospho-RTK Array Kit (R&D Systems) was used according to the manufacturer's instructions. Bands and dots were detected using an ImageQuant LAS-4000 imager (GE Healthcare Biosciences). Mean pixel density was measured using ImageJ (version 1.52a, National Institute of Health).
8
Growth Factor Signaling in Irradiated Cells
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Cells were seeded in complete medium in 10 cm fibronectin-coated tissue cultures dishes at a density of 5,000 cells/cm2 and allowed to reach confluence. Cells received either a 0 Gy (control) or 10 Gy (treatment) dose of radiation. Media was replaced with fresh complete media and cells incubated for a further 48 h. Cells were washed with serum-free medium and serum starved for 4 h prior to stimulation with a growth factor mixture consisting of VEGF-A (50 ng/mL for 5 min), VEGF-C (200 ng/mL for 10 min) or VEGF-D (200 ng/mL for 10 min), EGF [100 ng/mL for 5 min, PromoCell (Germany)], bFGF [100 ng/mL for 5 min, (PromoCell, Germany)], IGF-1 [100 nM for 5 min, (PromoCell, Germany)] and heparin (1 μg/mL for 5 min; added 5 min before the addition of bFGF). Cells were lysed and analysed with the human Phospho RTK-array kit (R&D Systems, United States) as per manufacturer’s instructions.
9
Western Blot Analysis of Phospho-RTK
10
Protein Extraction and Immunoblotting
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Proteins were extracted from cultured cells or concentrated FBS-free media using RIPA buffer (150 mM NaCl, 50 mM Tris, 1% Nonidet P-40, pH 7.5) supplemented with protease and phosphatase inhibitors (Roche) and boiled with SDS loading buffer for 10 minutes. The denatured proteins were resolved in a 4% to 20% SDS-PAGE gel for immunoblotting analysis with the indicated antibodies listed in Supplemental Table 10 . The blotted bands were visualized on X-ray films or by the Bio-Rad ChemiDoc XRS+ Imaging System. The Human Phospho-RTK Array Kit was purchased from R&D Systems.
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