Ipatasertib

Manufactured by Selleck Chemicals

Sourced in United States

Ipatasertib is a small-molecule inhibitor of the protein kinase AKT. It functions by blocking the activation of the AKT signaling pathway, which is involved in cellular processes such as cell growth, proliferation, and survival.

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Ipatasertib (GDC-0068) (6 citations)

19 protocols using ipatasertib

Pharmacological Inhibitor Acquisition Protocol

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The AKT inhibitors ipatasertib (#S2808), AZD5363 (#S8091), MK2206 (#S1078), SC66 (#S5313), and the BH3 mimetic ABT-737 (#S1002) were purchased from SelleckChem (Houston, TX, USA). ARQ-092 (#21388), perifosine (#1008112), PHT-427 (#24188), staurosporine (#81590), digitonin (#14952), and FCCP (#15218) were purchased from Cayman Chemical. Oligomycin (#O4876), β-mercaptoethanol (#M3148), and endoxifen (E8285) were purchased from Sigma Aldrich (St Louis, MO, USA).

Bougen-Zhukov N., Nouri Y., Godwin T., Taylor M., Hakkaart C., Single A., Brew T., Permina E., Chen A., Black M.A, & Guilford P. (2019). Allosteric AKT Inhibitors Target Synthetic Lethal Vulnerabilities in E-Cadherin-Deficient Cells. Cancers, 11(9), 1359.

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Cell Adhesion Assay for Vitronectin

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Cell adhesion assays were performed using 24-well tissue culture plates uncoated/coated with vitronectin (VN) (Corning, NY 14831 USA). In details, plates were incubated ON with VN (2,5 μg/mL in PBS) at 4 °C. After gentle washing with PBS, 1 h BSA1% incubation at RT and again washing with PBS, recipient cells treated with either PBS or LO (20 μg/mL) were plated in each well (6 × 10⁴ viable cells/well) and incubated at 37 °C, 5% CO2 in serum-free medium. When indicated, the adhesion assay was preceded by a pre-treatment: LO pre-treated for 45 min with 1:500 diluted blocking anti-αV monoclonal antibody, clone 272-17E6 (Merck Millipore, Burlington, Massachusetts, USA); or recipient cells with GDC-0068 AKT inhibitor (Ipatasertib by Selleckchem, Munich, Germany). Dosage and time of treatment as indicated in figures. As negative controls for either anti-αV-pre-treated LO, or GDC-0068 pre-treated recipient cells, cells were treated with anti-αV monoclonal blocking antibody or AKT inhibitor alone, respectively. At the indicated times, the adherent cells were counted and arbitrary values of 100% was given to the basal adhesion of cells treated with only PBS and values were reported relative to that.

Ciardiello C., Leone A., Lanuti P., Roca M.S., Moccia T., Minciacchi V.R., Minopoli M., Gigantino V., De Cecio R., Rippa M., Petti L., Capone F., Vitagliano C., Milone M.R., Pucci B., Lombardi R., Iannelli F., Di Gennaro E., Bruzzese F., Marchisio M., Carriero M.V., Di Vizio D, & Budillon A. (2019). Large oncosomes overexpressing integrin alpha-V promote prostate cancer adhesion and invasion via AKT activation. Journal of Experimental & Clinical Cancer Research : CR, 38, 317.

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HNSCC Cell Culture and Drug Treatment

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HNSCC FaDu cell line was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Authenticity of the cells was confirmed by short tandem repeat profile. The cells were maintained in a humidified atmosphere of 5% CO₂ at 37 °C in RPMI-1640 medium (Sigma-Aldrich Corp., St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. Cetuximab, ipatasertib, afatinib, and dacomitinib were obtained from Selleck Chemicals (Houston, TX, USA).

Yonesaka K., Tanaka K., Kitano M., Kawakami H., Hayashi H., Takeda M., Sakai K., Nishio K., Doi K, & Nakagawa K. (2019). Aberrant HER3 ligand heregulin-expressing head and neck squamous cell carcinoma is resistant to anti-EGFR antibody cetuximab, but not second-generation EGFR-TKI. Oncogenesis, 8(10), 54.

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Elucidating Splicing Regulation by TCR Signaling

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To elucidate the roles of signal transduction proteins to regulate splicing changes, each protein known to be regulated downstream of TCR signaling was targeted by at least two different inhibitors. Human primary CD4+ CD45RO- T cells were incubated with individual inhibitors for 1 hr and stimulated with bound anti-CD3 and soluble anti-CD28 antibodies for 48 hr. Cells were then harvested for RNA purification and low-cycle RT-PCR analysis for splicing quantifications. The inhibitors utilized are as follows: PCKi: R0-31-8220 (Selleckchem: S7207) and Go6983 (Selleckchem: S2911), p38i: SB20350 (Selleckchem: S1076) and Skepinone-L (Selleckchem: S7214), NFATi: Cyclosporin A (Selleckchem: S2286) and FK506 (Selleckchem: S5003), AKTi: MK-2206 (Selleckchem:S1078) and Ipatasertib (Selleckchem:S2808), JNKi: SP600215 (Selleckchem: S1460) and JNK-IN-8 (Selleckchem: S4901) and Tanzisertib (Selleckchem: S8490).

Blake D., Radens C.M., Ferretti M.B., Gazzara M.R, & Lynch K.W. (2022). Alternative splicing of apoptosis genes promotes human T cell survival. eLife, 11, e80953.

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Cardiomyocyte Hypertrophy Induction Protocol

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To induce cardiomyocyte hypertrophy, the cardiomyocytes were starved with 1% FBS for 24 h. Then, the cardiomyocytes were subjected to hypertrophy induction with ISO (50 μM) treatment for 48 h. A-actinin staining was performed to stain and indicate cardiomyocytes, then cell size was analyzed with the ImageJ software. For drug treatment, rapamycin, LY294002, and Ipatasertib were purchased from Selleck and the concentrations were indicated in the figure legends.

Gao W., Guo N., Zhao S., Chen Z., Zhang W., Yan F., Liao H, & Chi K. (2020). Carboxypeptidase A4 promotes cardiomyocyte hypertrophy through activating PI3K-AKT-mTOR signaling. Bioscience Reports, 40(5), BSR20200669.

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Cell Signaling Pathway Analysis Protocol

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Abemaciclib, BYL719, buparlisib, AZD8186, ipatasertib, AZD5363, and MK2206 were purchased from Selleckchem (Houston, TX, USA). Antibodies against the following proteins were purchased from Santa Cruz Biotechnology (Dallas, TX, USA): p107 (sc-250), p130 (sc-9963), cyclin A (sc-751), cyclin D1 (sc-753), cyclin E (sc-481), CDK2 (sc-163), and GAPDH (sc-47724). Antibodies against Rb (cs#9309), p-Rb (cs#8180), E2F (cs#3742), p16 (cs#92803), p-AKT S473 (cs#4058), p-AKT T308 (cs#9275), AKT (cs#4685), p-TSC2 (cs#3617), TSC2 (cs#4308), and vinculin (cs#13901) were purchased from Cell Signaling Technology (Danvers, MA, USA). Recombinant protein human epithelial growth factor (rhEGF) and recombinant protein human fibroblast growth factor (rhEGF) were purchased from R&D Systems (Minneapolis, MN, USA). Mitomycin C (MMC), propidium iodide (PI), RNase, and sodium dodecyl sulfate (SDS) were purchased from Sigma Aldrich (St. Louis, MO, USA). Phosphate buffered saline (PBS) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Roswell Park Memorial Institute (RPMI) 1640 and Dulbecco’s modified Eagle’s medium (DMEM) medium were purchased from Welgene (Daejeon, Korea). All chemicals and reagents were of analytical grade and were obtained from commercial sources.

Lee H.J., Kim K.J., Sung J.H., Nam M., Suh K.J., Kim J.W., Kim S.H., Kim J.W., Kim Y.J., Lee K.W., Lee J.S, & Kim J.H. (2020). PI3K p110α Blockade Enhances Anti-Tumor Efficacy of Abemaciclib in Human Colorectal Cancer Cells. Cancers, 12(9), 2500.

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Pharmacological Inhibition of SGK1

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pBABE–puro retroviral vector was purchased from Addgene. Ipatasertib, MK-2206, temsirolimus, SGK233470, wortmannin, rapamycin, and SGK650394 were purchased from Selleckchem. SGK1 inhibitors compound 14g and mp-1 were provided by Dr. Marc Nazare (Leibniz-Forschungsinstitut fϋr Molekulare Pharmakologie (FMP)). Unless specified, all in vitro treatments were performed using 1μM concentration for 3 hours.

Slotkin E.K., Diolaiti D., Shukla N.N., Cruz F.S., Clark J.J., Gundem G., Yellapantula V.D., Levine M.F., You D., Ma P., Pachhal S., Sanchez G.I., Benayed R., Jungbluth A.A., Smyth L.M., Mauguen A., Gushterova I., Ding H., Spraggon L., Darnell R., Califano A., Ladanyi M., Papaemmanuil E., Kung A.L., Hyman D.M, & Roberts S.S. (2019). Patient-driven Discovery, Therapeutic Targeting, and Post-Clinical Validation of a Novel AKT1 Fusion-driven Cancer. Cancer discovery, 9(5), 605-616.

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Kinase Inhibitor Signaling Pathways

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A series of well characterized kinase inhibitors, including U0126 (Promega), SB203580 (Selleckchem), Ipatasertib (Selleckchem) and SP600125 (Selleckchem) were used to target MEK1/2, p38/MAPK, AKT and JNK signaling pathways, respectively. Inhibitors were supplemented into MCF10A cell culture medium individually after serum deprivation and also after serum replenishment for 30 minutes, 1 hour, 3 hours and 6 hours, all at the concentration of 10 μM. Cells were further collected for both RT-qPCR assay and NR4A1 ChIP-qPCR assays to assess IEG gene expression changes and NR4A1 genebody localization alterations upon serum stimulation.

Guo H., Golczer G., Wittner B.S., Langenbucher A., Zachariah M., Dubash T.D., Hong X., Comaills V., Burr R., Ebright R.Y., Horwitz E., Vuille J.A., Hajizadeh S., Wiley D.F., Reeves B.A., Zhang J.M., Niederhoffer K.L., Lu C., Wesley B., Ho U., Nieman L.T., Toner M., Vasudevan S., Zou L., Mostoslavsky R., Maheswaran S., Lawrence M.S, & Haber D.A. (2021). NR4A1 regulates expression of immediate early genes, suppressing replication stress in cancer. Molecular cell, 81(19), 4041-4058.e15.

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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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Modulation of S100A8/A9 Signaling Pathways

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Recombinant Human S100A8/A9 Heterodimer (carrier-free) (#753406) was purchased from Biolegend® (San Diego, CA). RAGE antagonist FPS-ZM1 (#S8185), AKT inhibitor Ipatasertib (#S2808), ROCK inhibitor Y-27632 2HCl (#S1049) and Myosin II inhibitor Blebbistatin (#S7099) were obtained from Selleckchem. FAK inhibitor VS-4718 (# HY-13917) and YAP/TEAD binding disruptor Verteporfin (# HY-B0146) were purchased from MedChemExpress (MCE). rhS100A8/A9 was diluted at 100 μg/mL in 1X PBS, while each pharmacological inhibitor was prepared as a 10 mM stock solution in DMSO.

Rigiracciolo D.C., Nohata N., Lappano R., Cirillo F., Talia M., Adame-Garcia S.R., Arang N., Lubrano S., De Francesco E.M., Belfiore A., Gutkind J.S, & Maggiolini M. (2022). Focal Adhesion Kinase (FAK)-Hippo/YAP transduction signaling mediates the stimulatory effects exerted by S100A8/A9-RAGE system in triple-negative breast cancer (TNBC). Journal of Experimental & Clinical Cancer Research : CR, 41, 193.

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