Mk 2206

Manufactured by Santa Cruz Biotechnology

Sourced in United States

MK-2206 is a laboratory reagent manufactured by Santa Cruz Biotechnology. It is a synthetic small molecule that functions as an allosteric inhibitor of the serine/threonine kinase AKT. The core function of MK-2206 is to inhibit the activity of AKT, which plays a crucial role in various cellular processes, including cell growth, proliferation, and survival.

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AKT inhibitor MK-2206 (2 citations)

22 protocols using mk 2206

Modulating PI3K/AKT and CDK Pathways

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To block PI3K/AKT activity, embryos were treated with LY294002 (M1925, Abmole) or MK-2206 (sc-364537, Santa Cruz) at indicated concentrations from 18 or 36 hpf to the desired stages. For AKT inhibition in cultured cells, HEK293T cells were treated with 0.5 μM MK-2206 for 24 h before harvest. To activate PI3K/AKT activity, embryos were treated with 1 μM 740 Y-P (1236188-16-1, R&D Systems) or 0.5 μM SC79 (SF2730, Beyotime) from 18 hpf until harvest, respectively, and HeLa cells were treated with 10 μM SC79 for 2 h before harvest. To block CDK activity, embryos were treated with 25 μM PD0332991 (A8318, Palbociclib) or 25 μM CY202 (A1723, Palbociclib) from 18 hpf until harvest. To examine which pathway was required for protein degradation, HEK293T cells expressing either Flag-tagged zEtv2 or zScl were treated with 20 mM NH4Cl (A116363, Aladdin) or 20 μM MG132 (M7449, Sigma) for 5 h before harvest.

Liu J., Zhang M., Dong H., Liu J., Mao A., Ning G., Cao Y., Zhang Y, & Wang Q. (2022). Chemokine signaling synchronizes angioblast proliferation and differentiation during pharyngeal arch artery vasculogenesis. Development (Cambridge, England), 149(23), dev200754.

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Regulation of AKT/mTOR Signaling Pathway

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RPMI 1640 and fetal bovine serum (FBS) were purchased from Welgene, Inc. (Daegu, South Korea). Lipofectamine™ 2000 Reagent was purchased from Invitrogen (Carlsbad, CA). D-erythro- and L-threo-SPC were purchased from Matreya (Pleasant Gap, PA). MK2206 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-AKT1 and phospo-specific antibodies to detect K8 S431 were supplied from Abcam (Cambridge, UK). Anti-RhebL1 antibody and peroxidase-labeled secondary antibodies were acquired from Santa Cruz Biotechnology. Anti-phospho-AKT Ser 473 was acquired from Cell Signaling Technology (Beverly, MA). Alexa Fluor 488 donkey anti-goat antibody and Alexa Fluor 594 chicken anti-rabbit antibody were obtained from Molecular Probes, Inc. (Eugene, OR).

Kim H.J., Byun H.J., Park M.K., Kim E.J., Kang G.J, & Lee C.H. (2017). Novel involvement of RhebL1 in sphingosylphosphorylcholine-induced keratin phosphorylation and reorganization: Binding to and activation of AKT1. Oncotarget, 8(13), 20851-20864.

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Immortalized Tubular Epithelial Cell Culture

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Immortalized tubular epithelial cells (HK-2) were cultured in DMEM/F12 medium supplemented with 10% FBS. After synchronization, cells were treated with 20% FSGS patients’ serum (PS) or 40 nM C3a (204881, Merck-Calbiochem). For intervention studies, 1 μM of C3aR antagonist SB290157 (sc-222291, Santa Cruz), 100 μg/ml of eculizumab (Soliris, Alexion Pharmaceuticals), 10 μM of p38 MAPK inhibitor SB203580 (sc-3533, Santa Cruz), 50 μM of ERK inhibitor PD98059 (sc-3532, Santa Cruz), 10 μM of Akt inhibitor MK2206 (sc-364537, Santa Cruz) or 1 μM of U-46619 (sc-201242, Santa Cruz) was given 30 min before treatments. To infect HK-2 cells with plenti-CMV-LOC105375913, plenti-CMV-snail or plenti-CMV-XBP-1s plasmid, the lentiviral stock was mixed with polybrene (1 µg/ml) and added to cells. C/EBPβ siRNA (sc-44251), Elk-1 siRNA (sc-35290), ERα siRNA (sc-29305), GR siRNA (sc-35505), snail siRNA(sc-38398) and XBP-1s siRNA (sc-38627) were bought from Santa Cruz (Dallas, Texas, USA). LOC105375913 siRNA was bought from Thermo fisher (4390771, Carlsbad, California, USA). Transfection of siRNA, miRNA mimics or miRNA antisense oligonucleotide (ASO) was conducted with Lipofectamine 2000.

Han R., Hu S., Qin W., Shi J., Zeng C., Bao H, & Liu Z. (2019). Upregulated long noncoding RNA LOC105375913 induces tubulointerstitial fibrosis in focal segmental glomerulosclerosis. Scientific Reports, 9, 716.

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Investigating Insulin Signaling Pathways

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SH-SY5Y human neuroblastoma cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mmol L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin in a 37 °C, 5% CO2 humidified atmosphere. Serum starved cells were then incubated for 4 h in the presence or absence of palmitic acid (200 µM), DHA (20 µM), resistin (200 ng/mL) or insulin (100 nM) and/or challenged for 15 min with insulin (100 nM), to evaluate the impact of fatty acids and resistin treatment on SH-SY5Y insulin responsiveness, or with resistin (200 ng/mL), to analyze the effect of fatty acid treatment on resistin/TLR4 signaling. MK-2206 (Akt inhibitor), U0126 (MEK1/2 inhibitor), SP600125 (JNK inhibitor), SB202190 (p38 MAPK inhibitor) were provided by Santa Cruz Biotechnology and were dissolved in DMSO for cell culture experiments, the control cells were treated with DMSO alone. Cells were exposed to 1 µM MK-2206, 10 µM U0126, 20 µM SP600125, 50 µM SB202190 for 1 h and then after washout cells were treated with resistin (200 ng/mL) for 4 h.

Amine H., Benomar Y, & Taouis M. (2021). Palmitic acid promotes resistin-induced insulin resistance and inflammation in SH-SY5Y human neuroblastoma. Scientific Reports, 11, 5427.

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Oral Administration of MK-2206 in Mice

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120 mg/kg body weight of MK-2206 (Santa Cruz Biotechnology, # 1032350-13-2) was administered orally. Captisol (Captisol) was used as a vehicle for the drug and the control animals were treated with vehicle only. MK-2206 was given orally for 3 weeks on alternate days. The dose and the duration mentioned in the study have been provided by Merck and Co.

Benichou E., Seffou B., Topçu S., Renoult O., Lenoir V., Planchais J., Bonner C., Postic C., Prip-Buus C., Pecqueur C., Guilmeau S., Alves-Guerra M.C, & Dentin R. (2024). The transcription factor ChREBP Orchestrates liver carcinogenesis by coordinating the PI3K/AKT signaling and cancer metabolism. Nature Communications, 15, 1879.

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Investigating AKT, PI3K, and SUMOylation Inhibitors

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All chemicals for the experiments were reagent grade or better. The AKT inhibitor, MK2206, was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The PI3K inhibitor, LY294002, was obtained from Cell Signaling Technology (Beverly, MA, USA). The SUMOylation inhibitor, ginkgolic acid, was purchased from Abcam (Cambridge, MA, USA). The protease inhibitor, MG132, was provided by Millipore (Billerica, MA, USA). Antibodies used in this study are listed in Supplementary Table 4. CD44 was detected with pan-CD44 antibodies [CD44-APC, CD44 (8E2)], which detect all CD44 isoforms (CD44s and various CD44v).

Kim I.G., Lee J.H., Kim S.Y., Heo C.K., Kim R.K, & Cho E.W. (2021). Targeting therapy-resistant lung cancer stem cells via disruption of the AKT/TSPYL5/PTEN positive-feedback loop. Communications Biology, 4, 778.

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Agar Preparation for Cell Cultures

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Low molecular weight (Ultra-agar Ina, average MW 43,000) and standard (S-6, average MW 290,000) agar was purchased from Ina Food Industry Co., Ltd. (Nagano, Japan). To prepare agar stock solution for cell cultures, agar was suspended in pure water to 1.0% (w/v) and dissolved by stirring at 90 °C. The aqueous solution was sterilized at 121 °C for 20 minutes by autoclave. The solution was then added to cell culture media at given concentrations with stirring at room temperature.
Trametinib and MK-2206 were obtained from Santa Cruz (Texas, USA). Paclitaxel was purchased from Wako Pure Chemical Industries (Osaka, Japan).

Abe-Fukasawa N., Otsuka K., Aihara A., Itasaki N, & Nishino T. (2018). Novel 3D Liquid Cell Culture Method for Anchorage-independent Cell Growth, Cell Imaging and Automated Drug Screening. Scientific Reports, 8, 3627.

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Preparation and Culture of Cortical Neurons

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Cgcs were prepared as described previously (58 (link)). Cells were kept in Neurobasal medium (Thermo Fisher Scientific) supplemented with 1% penicillin/streptomycin/glutamine (Sigma-Aldrich), 2% B-27 (Thermo Fisher Scientific), and an additional 20 mM KCl. Coverslips and dishes were coated with poly-l-ornithine or poly-l-lysin (2 hours to overnight; Sigma-Aldrich), and for coverslips laminin (10 μg/ml; Sigma-Aldrich) was subsequently added for 2–3 hours at 37°C. MK-2206 (Santa Cruz Biotechnology) was added 2–3 hours after preparation.

zur Nedden S., Eith R., Schwarzer C., Zanetti L., Seitter H., Fresser F., Koschak A., Cameron A.J., Parker P.J., Baier G, & Baier-Bitterlich G. (2018). Protein kinase N1 critically regulates cerebellar development and long-term function. The Journal of Clinical Investigation, 128(5), 2076-2088.

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VEGF-A Knockdown and AKT Inhibition

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Exponential growth phase cells were plated in 6-well plates at a density of 0.5×10⁵ cells/ml and cultured for 24 hours before experimentation.
Expression of murine VEGF-A was knocked down with small interfering RNA (siRNA) duplexes specifically targeted to murine VEGF-A mRNA. Transfection of siRNA was performed using Oligofectamine (Invitrogen, Carlsbad, CA). The target sequences for VEGF-A mRNA were: 5′-TGCTGTGAAGATGTACTCTATCTCGTGTTTTGGCCACTGACTGACACGAGATAGTACA-TCTTCA-3′, 5′-CCTGTGAAGATGTACTATCTCGTGTCAGTCAGTGGCCAAAACC-3′. Non-targeting siRNA pool (D-001206-13-05; Dharmacon, Fisher Scientific, Pittsburgh, PA) was used as a negative control. Cells were transfected with 1 µg of siRNA in reduced serum medium (OPTI-MEM-I; Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol and harvested 72 hours post transfection. The RNA and protein were extracted and analyzed, respectively. For the AKT inhibition assay, cells were treated with vehicle, dimethyl sulfoxide (DMSO) or 100 nM MK-2206 (sc-364537, Santa Cruz, CA, USA) for 72 hours and then harvested for immunofluorescence staining and Western blot analysis.

Bai X., Li X., Tian J, & Zhou Z. (2014). Antiangiogenic Treatment Diminishes Renal Injury and Dysfunction via Regulation of Local AKT in Early Experimental Diabetes. PLoS ONE, 9(4), e96117.

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Pharmacological Formulation Guidelines

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The following drugs were used in the present studies: AR-A014418, SB216763 and U0126 were purchased from Tocris (Minneapolis, MN). Perifosine, nifedipine and sulpride were obtained from Sigma Sigma/RBI (St. Louis, MO). MK-2206 was acquired from Santa Cruz Biotechnology (Dallas, TX). The serotonin 5-HT₃R antagonist palonosetron and the NK₁R antagonist netupitant were kindly provided by Helsinn Health Care (Lugano, Switzerland).
Palonosetron was dissolved in water. MK-2206 and AR-A014418 and were dissolved in 25% DMSO in water. SB216763 was dissolved in 2.5% DMSO in water. Sulpride was dissolved in distilled water with a 10 μl volume of 1/3 concentrated HCl which was then back titrated to pH 5 by the addition of NaOH. Nifedipine, netupitant and U0126 were dissolved in a mixture of ethanol/Tween 80/saline at a volume ratio of 1:1:18 so that administration of large amounts of DMSO to the shrews could be avoided. All drugs were administered at a volume of 0.1 ml/10 g of body weight.

Zhong W., Chebolu S, & Darmani N.A. (2021). Central and peripheral emetic loci contribute to vomiting evoked by the Akt inhibitor MK-2206 in the least shrew model of emesis. European journal of pharmacology, 900, 174065.

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