Human recombinant leptin, human VEGFR-2 Quantikine ELISA Kits, and human VEGF cytokine (293-VE) was purchased from R&D Systems, Minneapolis, MN. Notch1 (sc-373891), Notch4 (sc-56594) and Jagged1 (JAG1, sc-8303) polyclonal antibodies were obtained from Santa Cruz Biotechnology, Santa Cruz, CA. DLL4 (ab7280), Notch2 (ab8926), and Notch3 (ab23426) polyclonal antibodies were purchased from Abcam, Cambridge, MA. VEGFR-2 monoclonal antibody (55B11) was purchased from Cell Signaling, Danvers, MA. Anti-mouse and anti-rabbit polyclonal antibodies conjugated to horseradish peroxidase were from Bio-Rad Laboratories, Hercules, CA. SU5416 was purchased from Sellekchem, Houston TX. Enhanced chemiluminescence (ECL)-western blot stripping buffer was from Thermo Scientific, Rockford, IL. Pegylated leptin receptor antagonist 2 (PEG-LPrA2) was prepared by us as previously described (Gonzalez and Leavis, 2003 (link)). Beta-actin (A5316) and GAPDH (glycerol aldehyde phosphate dehydrogenase; G8795) monoclonal antibodies, protease and phosphatase inhibitor cocktails 1 and 2, fetal bovine serum (FBS), DAPT [N-[N-(3,5-difluorophenacetyl)-l -alanyl]-S-phenylglycine t-butyl ester], 4’,6-diamidino-2-phenylindole] and S2188 gamma-secretase inhibitors, and other chemicals were purchased from Sigma-Aldrich, St. Louis, MO.
Su5416
Manufactured by Selleck Chemicals
Sourced in United States
SU5416 is a selective inhibitor of the vascular endothelial growth factor receptor (VEGFR) tyrosine kinase. It acts by blocking the signal transduction pathways associated with VEGFR, which are involved in the regulation of angiogenesis.
Automatically generated - may contain errors
Lab products found in correlation
Protease and phosphatase inhibitor cocktails 1 and 2, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Recombinant human leptin, by R&D Systems (1 mentions)
Beta actin a5316, by Merck Group (1 mentions)
Gapdh, by Merck Group (1 mentions)
Zd6474, by Selleck Chemicals (1 mentions)
Gremlin, by Merck Group (1 mentions)
Axitinib, by Selleck Chemicals (1 mentions)
Mk 2206, by Selleck Chemicals (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Okadaic acid, by Enzo Life Sciences (1 mentions)
6 protocols using su5416
1
Notch Signaling Pathway Inhibition
2
Stereotactic Injection of VEGF and SU5416 in Rat Seizure Model
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The rats were anesthetized via ether inhalation and placed in a stereotactic frame. The anterior fontanelle was used as the origin. A Hamilton syringe was placed in the right lateral cerebral ventricle at predetermined coordinates: 1.2 mm posterior to and 1.2 mm lateral from lambda and a depth of 3.8 mm. A drill was used to create a small hole in the skull, a catheter was inserted (Roanoke Corporation, USA) into the lateral ventricle and fixed with glass ionomer cement, and a tube was inserted into the catheter to seal it after the cement solidified. After the operation was completed, the rats were placed in cages and kept warm. After the operation, an i.p. injection of 300,000 units/kg penicillin was administered to prevent infection.
Rats with SE were randomly divided into the SV0, SU0, SV5, and SU5 groups and received injection of VEGF 165 40 ng [8 ng/μl, dissolved in phosphate-buffered saline (PBS), Peprotech, Rocky Hill, USA] or SU5416 5 mM [1 mM/μl, dissolved in dimethylsulfoxide (DMSO), Selleck, USA] per rat for 3 continuous days (2 h, 1 d, and 2 d or 5 d, 6 d, and 7 d after SE) at a rate of 0.5 μl/min. After injection, the microsyringe was kept in place for 5 min until the liquid was fully diffused. Then, the injection needle was removed, and the cap was closed.
Rats with SE were randomly divided into the SV0, SU0, SV5, and SU5 groups and received injection of VEGF 165 40 ng [8 ng/μl, dissolved in phosphate-buffered saline (PBS), Peprotech, Rocky Hill, USA] or SU5416 5 mM [1 mM/μl, dissolved in dimethylsulfoxide (DMSO), Selleck, USA] per rat for 3 continuous days (2 h, 1 d, and 2 d or 5 d, 6 d, and 7 d after SE) at a rate of 0.5 μl/min. After injection, the microsyringe was kept in place for 5 min until the liquid was fully diffused. Then, the injection needle was removed, and the cap was closed.
3
Pulmonary Hypertension Animal Model
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Sprague-Dawley rats (n = 6 in each group) were given a single i.p. injection of Sugen5416
(SU5416, 20 mg/kg, Selleck, USA) in vehicle (0.5% carboxyl methylcellulose sodium, 0.4%
polysorbate 80, 0.9% benzyl alcohol), placed immediately into a 10% O2 chamber
and maintained in hypoxia for three weeks. C57 mice (8–10 weeks, n = 6 in each group) were
given a single weekly i.p. injection of Sugen5416, placed immediately into a 10%
O2 chamber and maintained in hypoxia for three weeks. After three weeks in
hypoxia, rats and mice were placed in a normoxic environment for five weeks to induce
pulmonary hypertension. The animals were anesthetized by intraperitoneal application of
sodium pentobarbital (50 mg/kg) and euthanized by CO2 inhalation after the
hemodynamic assessment.
(SU5416, 20 mg/kg, Selleck, USA) in vehicle (0.5% carboxyl methylcellulose sodium, 0.4%
polysorbate 80, 0.9% benzyl alcohol), placed immediately into a 10% O2 chamber
and maintained in hypoxia for three weeks. C57 mice (8–10 weeks, n = 6 in each group) were
given a single weekly i.p. injection of Sugen5416, placed immediately into a 10%
O2 chamber and maintained in hypoxia for three weeks. After three weeks in
hypoxia, rats and mice were placed in a normoxic environment for five weeks to induce
pulmonary hypertension. The animals were anesthetized by intraperitoneal application of
sodium pentobarbital (50 mg/kg) and euthanized by CO2 inhalation after the
hemodynamic assessment.
4
Antibody and Inhibitor Procurement
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Gremlin and anti-b-actin monoclonal antibody were obtained from Sigma (Shanghai, China). VEGFR2 inhibitors SU5416, ZD6474 and Axitinib as well as the Akt specific inhibitor MK-2206 were obtained from Selleck (Nanjing, China). All other antibodies utilized in this study were obtained from Cell Signaling Technology (Nanjing, China).
5
Generating Metastatic Tumor Cells from B16 Melanoma
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Mouse B16-F10 cells were purchased from Wuhan Boster Biology Technology, Ltd. (Wuhan, China); mouse B16-F1 cells and mouse 3T3 cells were from China Center for Type Culture Collection (Wuhan, China). tfRFP-expressing B16-F10 cells were obtained by stably transfecting B16-F10 cells with a plasmid containing the KatushkaS158A gene17 (link). B16-F1, tfRFP B16-F10, and 3T3 cells were cultured on rigid dishes with MEM, 1640 and DMEM cell culture medium (HyClone) respectively supplemented with 10% fetal bovine serum (Invitrogen), and 1% penicillin/streptomycin at 37 °C with 5% CO2. Cells were passaged every 2–3 days using TrypLE (Invitrogen). TRCs were mechanically selected by being cultured in the 3D soft fibrin gels (90-Pa) for 5 days12 (link) from B16 cell lines and both were more metastatic than their respective counterpart control cells. Dexamethasone (Dex), Latrunculin A (Lat A), retinoic acid (RA), dimethyl sulfoxide (DMSO), and ethanol were from Sigma. Angiogenic and vasculogenic blocker SU5416 was purchased from Selleck. Its stock solution was 10 mM in DMSO. The drug was diluted 5000 times such that its final working concentration was 2 μM with 0.02% DMSO in fish medium. After 1 hr incubation with the drug, fresh medium was added to wash out the drug. The drug-treated embryos appeared slightly yellowish, suggesting good absorption.
6
Embryonic Heat Shock and Chemical Inhibition
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Heat shock was performed at the 13-somite stage (ss) of embryonic development for 45 min in a 39°C water bath. Embryos were returned to 1xE3 (room temperature) and incubated at 28°C. Subsequent chemical inhibitor treatments were performed 0.5-1 hour after the heat shock and incubated for the stated times at 28°C in the dark. The chemicals were diluted in 1xE3 in the following concentrations: 1.5µM of the VEGFR2 inhibitor SU5416 (Selleckchem) and 15nM Okadaic acid (Enzo).
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