Azd1480

Manufactured by Selleck Chemicals

Sourced in United States

AZD1480 is a small molecule that functions as a tyrosine kinase inhibitor. It is used as a research tool to study cellular signaling pathways involving tyrosine kinases.

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Close spelling variants of this product

JAKi AZD1480 (2 citations)

52 protocols using azd1480

Evaluating AKTIP Inhibition in MCF7 Cells

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MCF7 cells stably expressing AKTIP shRNA or vector were seeded into 96-well plates in triplicate at the density of 1,000 cells per well 24 hrs before the addition of indicated pharmacological inhibitors. Fulvestrant and the JAK2 inhibitor AZD1480 were obtained from Selleckchem (Houston, TX) whereas 4-OH-Tam was obtained from APExBIO. Cells were treated with the inhibitors at serially diluted concentrations for indicated amount of time. DMSO was used as control. Cell viability was measured using resazurin.

Ng A.S., Zhang S., Mak V.C., Zhou Y., Yuen Y., Sharma R., Lu Y., Zhuang G., Zhao W., Pang H.H, & Cheung L.W. (2022). AKTIP loss is enriched in ERα-positive breast cancer for tumorigenesis and confers endocrine resistance. Cell reports, 41(11), 111821.

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Molecular Mechanisms of JAK2-FOXO3 Pathway

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Mouse anti-β-actin, mouse anti-Flag, and mouse anti-HA antibody and the following chemicals and solvents (MG132, cycloheximide, dimethyl sulfoxide (DMSO), glycerol, glycine, sodium chloride, Trizma base, and Tween20) were from Sigma (St. Louis, MO, USA). AZD1480 was from Selleckchem (Houston, TX, USA). Rabbit anti-IL4Rα, rabbit anti-IL13Rα1, mouse anti-PARP1, rabbit anti-FOXO3, mouse anti-Lamin B1, and mouse anti-GAPDH antibodies were from Santa Cruz Biotechnology. Rabbit anti-JAK2, rabbit anti-pJAK2, rabbit anti-Tyr, rabbit anti-cleaved PARP1, rabbit anti-cleaved Caspase3, rabbit anti-Bax, rabbit anti-Bim, rabbit anti-Bcl2, rabbit anti-p21, and rabbit anti-p27 antibodies were from Cell Signaling (Danvers, MA, USA). Goat anti-rabbit (111-035-003) and goat anti-mouse (115-035-003) horseradish peroxidase-conjugated IgG were obtained from Jackson ImmunoResearch (West Grove, PA, USA). Enhanced chemiluminescence (ECL) reagents were obtained from Genedepot (Barker, TX, USA). pECE empty/Flag-FOXO3 and pCMV3-C-HA empty/HA-JAK2 plasmid DNA were from Addgene (Watertown, MA, USA) and Sino Biological (Wayne, PA, USA), respectively.

Kang M.A., Lee J., Ha S.H., Lee C.M., Kim K.M., Jang K.Y, & Park S.H. (2019). Interleukin4Rα (IL4Rα) and IL13Rα1 Are Associated with the Progress of Renal Cell Carcinoma through Janus Kinase 2 (JAK2)/Forkhead Box O3 (FOXO3) Pathways. Cancers, 11(9), 1394.

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Sialidase-Modulated Platelet-Induced STAT3 Phosphorylation

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HepG2 cells (purchased from ATCC-HB-8065) were grown at 4×10⁵ cells/cm² in Permanox® (Nalge Nunc International) slides for 24 h in the presence of 2 μM AZD1480 or 10 μM of TG101348 (both Selleckchem) or 2 μM of BMS911543 (Chemie Tek) or vehicle before addition of human platelets treated or non-treated with sialidase. Immunofluorescence for anti-pSTAT3 (Cell Signaling Technology) was performed 1 h after platelet addition to HepG2 cells following antibody manufactor’s protocol. After 6 h incubation with humans platelets treated or non-treated with sialidase, HepG2 cells were fixed in 4% PFA, followed by permeabilization in 2% Triton X-100. Cells were incubated for 30 min at 37 °C with 5% goat-serum to block unspecific binding and incubated overnight at 4 °C with a polyclonal anti-human TPO antibody (R&D Systems, USA) followed by secondary AlexaFluo®568 conjugated polyclonal goat-anti rabbit antibody (Invitrogen). Nuclei were visualized by inclusion of 4′, 6-diamino-2-phenyllidole (DAPI) (Invitrogen) in the mounting medium.

Grozovsky R., Begonja A.J., Liu K., Visner G., Hartwig J.H., Falet H, & Hoffmeister K.M. (2014). The Ashwell-Morell receptor regulates hepatic thrombopoietin production via JAK2-STAT3 signaling. Nature medicine, 21(1), 47-54.

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Keratinocyte Serum-Free Culture Protocol

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Keratinocyte serum-free medium (KSFM), all the other medium power, Trizol reagent, Lipofectamine 2000, OPTI-MEM, anti-Myc epitope antibodies and zeocin were from Life Technologies (Grand Island, NY., USA). Endothelial growth medium 2 (EGM2) for growing endothelial cells was from Lonza Inc. (Allendale, NJ, USA). All the chemicals, anti-His tag antibodies and Wortmanin were from Sigma-Aldrich Co. (St. Louis, MO, USA). CellTiter 96^® AQueous One Solution (MTS kit) was from Promega Corp (Madison, WI, USA). pLKO_AS2.zeo was from National RNAi Core facility in Academia Sinica, Taiwan. Anti-CD15, anti-CD68, and anti-CD11b antibodies were from Leica Biosciences (Richmond, Illinois, USA). S100A9 antibodies and STAT3 inhibitor VI (iSTAT3) were from Santa Cruz Biotechnology (Dallas, Texas, USA). Anti-human CD34 antibody was from DakoCytomation Denmark A/S (Copenhagen, Denmark). Anti-S100A8 antibodies, human recombinant S100A9 (recS100A9) protein and anti-CD31 antibodies were from Abcam (Cambridge, MA, USA). Matrigel was from BD Biosciences (San Jose, CA, USA). Pyrrolidine dithiocarbamate (PDTC) was from Tocris Bioscience (Bristol, UK). AZD1480 was from Selleckchem (Houston, TX, USA).

Fang W.Y., Chen Y.W., Hsiao J.R., Liu C.S., Kuo Y.Z., Wang Y.C., Chang K.C., Tsai S.T., Chang M.Z., Lin S.H, & Wu L.W. (2015). Elevated S100A9 expression in tumor stroma functions as an early recurrence marker for early-stage oral cancer patients through increased tumor cell invasion, angiogenesis, macrophage recruitment and interleukin-6 production. Oncotarget, 6(29), 28401-28424.

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Browning Assays of JAK3 and SYK Inhibitors

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Following screening and hit validation, browning assays were performed with commercially available JAK3 inhibitor (tofacitinib, Selleckchem S5001) and SYK inhibitor (R406, Selleckchem S1533). PSC-WA and ADSCs were differentiated and treated following the assay design of the browning screen. tofacitinib was added at 2 μM and R406 at 1 μM final concentration unless otherwise indicated on figures and legends. THRB agonist (CAS 219691-94-8) was added at 5 μM final concentration. Ruxolitinib (Selleckchem S1378), Baricitinib (Selleckchem S2851), CYT387 (Selleckchem S2219) and AZD1480 (Selleckchem S2162) were added at 0.1-5 μM final concentration as indicated on figures.
Recombinant human BMP7 (R&D systems, 354-BP) was added at 10 ng/ml final concentration in adipogenic medium supplemented with 250 μM IBMX (3-Isobutyl-1-methylxanthine, Sigma-Aldrich I7018) at day 0, 3 and 5. In ADSCs, BMP7 was added at 10 ng/ml at day 7, 10 and 12. tofacitinib and R406 were added following the same conditions as BMP7 for proper comparison.

Moisan A., Lee Y.K., Zhang J.D., Hudak C.S., Meyer C.A., Prummer M., Zoffmann S., Truong H.H., Ebeling M., Kiialainen A., Gérard R., Xia F., Schinzel R.T., Amrein K.E, & Cowan C.A. (2014). White-to-brown metabolic conversion of human adipocytes by JAK inhibition. Nature cell biology, 17(1), 57-67.

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Browning Assays of JAK3 and SYK Inhibitors

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Evaluating Combination Therapies for Vemurafenib-Resistant Thyroid Cancer

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Vemurafenib, Fludarabine (STAT1 activation inhibitor) (Selleck Chemicals, #S1491) and AZD1480 (JAK2 inhibitor) (Selleck Chemicals, #S2162) were dissolved in DMSO, and stock solutions were stored at −80°C. 8505C, WRO, and their respective vemurafenib-resistant clonal cells were seeded in triplicate at a density of 10,000 cells per well, within 200 μl of phenol-free RPMI media (ThermoFisher) per well in 96-well flat-bottomed culture dishes. Cells were exposed to experimental drug regimens at the indicated concentrations after 24 hours and then incubated for 72 hours. Cell viability and apoptisis were assessed using the Vybrant ^® MTT Cell Proliferation Assay Kit (ThermoFisher #V13154) and Annexin V apoptosis detection kit (ThermoFisher #88–8007-72) according to manufacturer’s specifications. Data was collected using Tecan Infinite M1000 Pro Microplate reader for viability assessment.

Limberg J., Egan C.E., Gray K.D., Singh M., Loewenstein Z., Yang Y., Riascos M.C., Al Asadi H., Safe P., El Eshaky S., Liang H., Ullmann T.M., Wang W., Li W., Zhang T., Xiang J., Stefanova D., Jin M.M., Zarnegar R., Fahey TJ I.I.I, & Min I.M. (2023). Activation of the JAK/STAT Pathway leads to BRAF inhibitor Resistance in BRAFV600E Positive Thyroid Carcinoma. Molecular cancer research : MCR, 21(5), 397-410.

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Inhibitors Modulate STAT3 and JAK2 Signaling

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5-Aza-2-deoxycytidine (5-AzaC) and cycloheximide (CHX) were purchased from Sigma-Aldrich (St Louis, MO, USA). MG132 was purchased from ApexBio. Inhibitors of STAT3 (S3I-201), JAK2 (AZD1480), and Src (PP2) were purchased from Selleckchem (Selleckchem Chemicals, Houston, TX, USA). The cytotoxicity of S3I-201, AZD1480, and PP2 was measured by the MTT method (Figure S1). Antibodies against STAT3 (79D7), pSTAT3^Y705 (D3A7), JAK2 (D2E12), pJAK2 (3771), Src (36D10), and α-tubulin (2144) were purchased from Cell Signaling; anti-LNX1 (NBP1-49975, Novus, Littleton, CO, USA); anti-pSrc (9A6, Millipore, Billerica, MA, USA); anti-pJAK2 (PJAK2-240AP, FabGennix, Frisco, TX, USA); anti-GAPDH (GTX100118, GeneTex, Hsinchu, Taiwan); anti-LDOC1 (2507C1a, Santa Cruz, Dallas, TX, USA) for immunoprecipitation and immunofluorescent assay (IFA); anti-LDOC1 (LS-B3527, LifeSpan, Providence, RI, USA) for immunohistochemistry (IHC) study, and anti-β-actin (AC-15, Novus).

Lee C.H., Yang J.R., Chen C.Y., Tsai M.H., Hung P.F., Chen S.J., Chiang S.L., Chang H, & Lin P. (2019). Novel STAT3 Inhibitor LDOC1 Targets Phospho-JAK2 for Degradation by Interacting with LNX1 and Regulates the Aggressiveness of Lung Cancer. Cancers, 11(1), 63.

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Kinase Inhibitor Compounds Preparation

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JAK Inhibitors (AZD1480, baricitinib, peficitinib, ruxolitinib and pacritinib), Fms-related receptor tyrosine kinase 3 (FLT3) inhibitors (gilteritinib, quizartinib, FF10101, G-749 and MRX-2843) and other kinase inhibitors including an inhibitor of interleukin 1 receptor associated kinase 1 (IRAK1) and IRAK4, (IRAK1/4 inhibitor), and the ros proto oncogene (ROS1) /anaplastic lymphoma kinase (ALK) inhibitor lorlatinib were purchased from Selleck Chemicals LLC. pacritinib was also provided by CTI BioPharma Corp. through a Materials Collaborative Research and Development Agreement (M-CRADA). Inhibitors were dissolved in DMSO at a stock concentration of 10 mM/mL, except for pacritinib at 5 mM/mL. All stocks were aliquoted and stored at − 20 °C.

Wu Y., Wang V, & Yarchoan R. (2024). Pacritinib inhibits proliferation of primary effusion lymphoma cells and production of viral interleukin-6 induced cytokines. Scientific Reports, 14, 4125.

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Kinase Inhibitors and Interferon-gamma Assay

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The following inhibitors were used: Crizotinib (PF-02341066) (Selleckchem S106), Ceritinib (LDK378) (Selleckchem S7083), Alectinib (CH5424802) (Selleckchem S2762), Lorlatinib (PF-6463922) (Selleckchem S7536), Selumetinib (AZD6244) (Selleckchem S1008), Trametinib (GSK1120212) (Selleckchem S2673), Tofacitinib Citrate (CP-690550) (Selleckchem S5001), AZD1480 (Selleckchem S2162), Rapamycin (Selleckchem S1039), LY294002 (Selleckchem S1105), JNK-IN-8 (Selleckchem S4901), Ro-31-8220 (Selleckchem 7207), AZD5363 (Selleckchem S8017), Ipatasertib (Selleckchem S2808), SCH772984 (Selleckchem S7101), Ulixertinib (Selleckchem S7854). BKM120 and Tepp-46 were kind gifts from Dr. Thomas Craig, NCI, Bethesda, Maryland, USA; recombinant human interferon-gamma, IFNγ (BioLegend 570208).

Rajan S.S., Amin A.D., Li L., Rolland D.C., Li H., Kwon D., Kweh M.F., Arumov A., Roberts E.R., Yan A., Basrur V., Elenitoba-Johnson K.S., Chen X.S., Puvvada S.D., Lussier Y.A., Bilbao D., Lim M.S, & Schatz J.H. (2019). The Mechanism of Cancer Drug Addiction in ALK-Positive T-Cell Lymphoma. Oncogene, 39(10), 2103-2117.

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