As703026

Manufactured by Selleck Chemicals

Sourced in United States

AS703026 is a lab equipment product. It is a chemical compound used in research and laboratory settings.

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Lab products found in correlation

7 protocols using as703026

Investigating MAPK and AKT Signaling

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OVMANA cells were plated (5×10⁵) after adherence media were replaced with fresh media containing either DMSO (control), URML-3881 at varying concentrations, R05126766 at 10 μM, AS703026 at 10 μM, or sorafenib at 10 μM (all drugs except URML-3881 were obtained from Selleck Chemicals). Cell lysis buffer (Cell Signaling, 9803S), supplemented with Halt protease and phosphatase inhibitors (Thermo Scientific, 1862209 and 1862495), was utilized for protein extraction. Animal tumor tissue was snap frozen and stored in liquid nitrogen. It was then thawed and mechanically homogenized in cell lysis buffer. Protein concentrations were determined using the DC Protein Assay (Bio-Rad, 5000111). Western blot analysis was performed as previously described [22] (link). Primary antibodies against p-ERK (4370), ERK (9102), p-MEK (2338), MEK 1/2 (4694), p-AKT (9271), AKT (9272), pPEA-15 (2776), and PEA-15 (2780) were all obtained from Cell Signaling Technology and used at the recommended dilutions.

Rowswell-Turner R.B., Rutishauser J.A., Kim K.K., Khazan N., Sivagnanalingam U., Jones A.M., Singh R.K, & Moore R.G. (2019). Novel Small Molecule MEK Inhibitor URML-3881 Enhances Cisplatin Sensitivity in Clear Cell Ovarian Cancer. Translational Oncology, 12(7), 917-924.

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Cell Viability Assay with Small Molecule Inhibitors

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Small molecule inhibitors were purchased from Tocris Biosciences (R&D Systems): PF-4708671, U 73122, GSK2334470, IPA3, SL0101-1 and XMD 8-92 or from Selleck Chemicals LLC (TX, USA): PF-562271, Enzastaurin (LY317615), AUY922 (NVP-AUY922), 17-AAG (Geldanamycin), PF-04929113 (SNX-5422), AZD6244 (Selumetinib), AT7867, CHIR-98014, LY2228820, BIX 02188, AS703026, PH-797804, SP600125, NU7441. All inhibitors were prepaed in DMSO at 100 mM. Cells were treated with inhibitors prepared in culture media where the final concentration of DMSO was 2% v/v. Vehicle control treatments consisted of culture media containing 2% v/v DMSO. Six days after treatment, cell survival was measured in comparison to vehicle controls using the CellTiter 96® Assay as per manufacturer instructions (MTS assay, Promega Corporation, WI, USA). Data were analyzed in GraphPad Prism® version 5.00 for Windows (GraphPad Software, CA, USA) to measure the log₁₀ of IC50 for each drug. For combination assays, drugs were added simultaneously. Heatmaps for sensitivities (-log₁₀ of IC50) were prepared using D-chip Analyzer software [49 (link)].

Al-Ejeh F., Miranda M., Shi W., Simpson P.T., Song S., Vargas A.C., Saunus J.M., Smart C.E., Mariasegaram M., Wiegmans A.P., Chenevix-Trench G., Lakhani S.R, & Khanna K.K. (2014). Kinome profiling reveals breast cancer heterogeneity and identifies targeted therapeutic opportunities for triple negative breast cancer. Oncotarget, 5(10), 3145-3158.

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Inhibition of Signaling Pathways in Melanoma

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Human melanoma cell lines: primary WM115 (VGP) and metastatic: WM266–4 (derived from metastatic site – right thigh skin). These cells lines feature the specific V600D mutation in the B-RAF gene, as well as express PTEN loss of function including hemizygous deletion and wild type for N-ras, c-KIT and CDK4. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics: penicillin and streptomycin. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂ in air. Cells were treated with inhibitors of: 1/AKT – MK-2206 (Selleck) at 2 μM concentration, 2/ MEK – AS-703026 (Selleck) at 10 μM concentration, 3/ PI3K – LY294002 (Cell Signalling TM) at 20 μM concentration, 4/ ERK1/2 – U0126 (Cell Signalling TM) at 10 μM concentration, and 5/ mTOR – everolimus (Selleck) at: 20 nM, 2 μM, 5 μM and 10 μM concentrations. The incubation time of melanoma cells with inhibitors were 24 and 48 h. Cells were obtained from the ESTDAB Melanoma Cell Bank (Tubingen, Germany).

Ciołczyk-Wierzbicka D., Zarzycka M., Gil D, & Laidler P. (2019). mTOR inhibitor Everolimus-induced apoptosis in melanoma cells. Journal of Cell Communication and Signaling, 13(3), 357-368.

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AML Cell Line Characterization Protocol

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All human AML cell lines were purchased directly from recognized repositories; Kasumi-1, MV-4-11, CMK, AML-193, M-07e, HL-60, ML-2, OCI-AML3, ME-1 and HEL from DSMZ (Braunschweig, Germany) and THP-1 from ATCC (Manassas, USA).
AZD6244, PD0325901, GSK1120212, CI-1040, TAK-733, AS703026, MEK162, cytarabine (cytosine arabinoside), daunorubicin hydrochloride (daunorubicin) and etoposide were obtained from Selleck chemicals (Houston, USA). ARRY-162 was purchased from ChemieTek (Indiana, USA). All compounds were dissolved in DMSO and stored at –20° C.

Smith A.M., Zhang C.R., Cristino A.S., Grady J.P., Fink J.L, & Moore A.S. (2019). PTEN deletion drives acute myeloid leukemia resistance to MEK inhibitors. Oncotarget, 10(56), 5755-5767.

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Comprehensive Melanoma Cell Line Protocol

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WM115, WM266-4, A375, SK-MEL28, SK-MEL2 and RMPI-7951 were purchased at ATCC; WM35, WM983A, WM983B, WM239A, WM3211 and Lu1205 at Coriell Institute and WM1346 at Wistar Institute. WM115, WM266-4, A375 and SK-MEL28 were cultured in DMEM/FBS 10% (v/v), SK-MEL2, 501mel and RPMI-7951 in RPMI-1640/FBS 10% (v/v), WM35, WM983A, WM983B, WM3211, WM1346 and Lu1205 in MCDB153 medium with 20% Leibovitz L-15 medium (v/v), 2% FBS heat inactivated (v/v), 5 μg/mL insulin and 1.68 mM CaCl2. Cell lines were authenticated for mutations in BRAF and NRAS by sequencing within the time frame of the experiments. PLX4032, SB590885, AZD6244, AS703026 and MK2206 were from Selleck Chemicals, Z-VAD-FMK and actinomycin D from Sigma-Aldrich, LY294002 from Calbiochem, G594 competitive inhibitor of AKT was provided by Genentech.

Delmas A., Cherier J., Pohorecka M., Medale-Giamarchi C., Meyer N., Casanova A., Sordet O., Lamant L., Savina A., Pradines A, & Favre G. (2015). The c-Jun/RHOB/AKT pathway confers resistance of BRAF-mutant melanoma cells to MAPK inhibitors. Oncotarget, 6(17), 15250-15264.

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Melanoma Cell Lines and Inhibitor Treatments

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Early passage human melanoma cell lines, YUKSI, YUSIK, YUSIT1, YUSIV,
YUGEN8 and YUVON, were provided by Dr Ruth Halaban (Yale University School of
Medicine), and were maintained in Opti-Mem with 5% FBS, GlutaMax, Penicillin and
Streptomycin. HEK293T, HT29, MCF7, T47D, NCI-H460 and MDA-MB-231 cell lines were
cultured in DMEM with 10% FBS. Mouse KRASmut/p53−/− NSCLC line
(T1) was kindly provided by Dr Tyler Jacks (Koch Institute for Integrative
Cancer Research). RAS and BRAF mutational status of the various lines is
specified in the figures. GFP-LC3B baculovirus infection was performed following
the manufacturer’s instructions (P36235; Life Technologies, Carlsbad, CA,
USA). Lentivirus production, target cell infection and selection were performed
as previously described.42 (link) Vemurafenib
(S1267; Selleck Chemicals, Houston, TX, USA) and AS703026 (S1475; Selleck
Chemicals) were dissolved in DMSO, and CQ (C6628; Sigma, St Louis, MO, USA) was
dissolved in PBS. Cells were treated with Raf/MAPK inhibitors for 48 h unless
otherwise specified.

Sanduja S., Feng Y., Mathis R.A., Sokol E.S., Reinhardt F., Halaban R, & Gupta P.B. (2016). AMPK promotes tolerance to Ras pathway inhibition by activating autophagy. Oncogene, 35(40), 5295-5303.

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Evaluating MEK and Tankyrase Inhibitors

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Cells were seeded and treated with the indicated dose of MEK inhibitor (AZD6244, GSK112012, AS703026, or BAY 86-9766) (Selleckchem, Houston, TX). After 24 h, the cells were harvested and evaluated with the Trypan blue exclusion assay. Additionally, DLD-1, HCT-8, DLD-1 isogenic cells (PI3K mutant: 353), and HCT116 isogenic cells (54, 240, and 241) were plated and treated with 5 μM NVP-TNKS656 (tankyrase inhibitor) and 1 µM GSK112012 (MEK inhibitor). After 48 h, the cell death rate was determined with the Trypan blue exclusion assay.

Moon J.H., Hong S.W., Kim J.E., Shin J.S., Kim J.S., Jung S.A., Ha S.H., Lee S., Kim J., Lee D.H., Park Y.S., Kim D.M., Park S.S., Hong J.K., Kim D.Y., Kim E.H., Jung J., Kim M.J., Kim S.M., Deming D.A., Kim K., Kim T.W, & Jin D.H. (2019). Targeting β-catenin overcomes MEK inhibition resistance in colon cancer with KRAS and PIK3CA mutations. British Journal of Cancer, 120(9), 941-951.

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