Okadaic acid

Manufactured by Merck Group

Sourced in United States, Germany, Spain

Okadaic acid is a natural compound isolated from certain marine organisms. It functions as a protein phosphatase inhibitor, which means it can inhibit the activity of specific enzymes involved in the regulation of cellular processes. This compound is often used as a research tool in laboratory settings.

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Okadaic acid (OA; (2 citations)

134 protocols using okadaic acid

Worm Culture and Okadaic Acid Treatment

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All strains were grown at 20 °C under standard procedures according to Brenner [34 (link)] and fed Escherichia coli OP50-1 culture for 28–30 hours from the L1 stage. After this time, the worms were moved to NGM control plates (2 g NaCl, 3 g KH₂PO₄, 0.5 g K₂HPO₄, 0.0085 g/mL cholesterol diluted in 1 mL of absolute ethanol, 30 g Bactoagar, and H₂O up to 1 L) or to glucose-supplemented plates (100 mM) previously seeded with E. coli OP50-1 and supplemented with 49 μM of 5-fluoro-2′-deoxyuridine (FUDR, Sigma-Aldrich). For okadaic acid treatment, nematodes were placed on NGM plates containing 100 mM glucose, and then, okadaic acid (Sigma-Aldrich) was added to these plates to a final concentration of 30, 60, 120 and 240 nM and allowed to dry for approximately 20 minutes. Then, E. coli OP50-1 was seeded onto each plate. Worms were exposed for 24 hours to this condition. Three independent experiments were performed with more than 30 worms for each experimental condition.

Franco-Juárez B., Mejía-Martínez F., Moreno-Arriola E., Hernández-Vázquez A., Gómez-Manzo S., Marcial-Quino J., Arreguín-Espinosa R., Velázquez-Arellano A, & Ortega-Cuellar D. (2018). A high glucose diet induces autophagy in a HLH-30/TFEB-dependent manner and impairs the normal lifespan of C. elegans. Aging (Albany NY), 10(10), 2657-2667.

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Selective Protein Knockdown in Cell Lines

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NIH3T3, HEK293T, PC6-3, and A549 cells were cultured and transfected as described (Strack et al., 2004 (link); Jin et al., 2010 (link)). siRNA-mediated knockdown of endogenous B56ε in NIH3T3 or knockdown of endogenous Fam13a in A549 cells was performed using Lipofectamine 2000 transfection reagent (Life Technologies, Gaithersburg, MD). The sequences for the sense and antisense strands of siRNAs are as follows: siGFP, 5′-gcaagcugacccugaaguucuu-3′; 5′-gaacuucagggucagcuugcuu-3′. siB56ε, 5′-ccuagugacagcaaugaauuu-3′; 5′ -auucauugcugucacuagguu-3′. siFam13a, 5′-ggagaacucuuagaaagaauu-3′; 5′-uucuuucuaagaguucuccuu-3′. Knockdown of B56s in PC6-3 cells was carried out as described (Strack et al., 2004 (link); Jin et al., 2011 (link)). For PP2A and Akt inhibition studies, NIH3T3 cells were treated with okadaic acid (50 nM, #O7885; Sigma-Aldrich, St. Louis, MO) and/or wortmannin (2 μM, #W1628; Sigma-Aldrich) or LY294002 (#L9908; Sigma-Aldrich) in DMEM supplemented with 0.5% bovine calf serum 1 d after transfection and harvested 15 h after drug administration. For leptomycin B treatment, NIH3T3 cells were exposed to 5 ng/ml leptomycin B (#L2913; Sigma-Aldrich) and/or 50 nM okadaic acid for 15 h before fixation.

Jin Z., Chung J.W., Mei W., Strack S., He C., Lau G.W, & Yang J. (2015). Regulation of nuclear–cytoplasmic shuttling and function of Family with sequence similarity 13, member A (Fam13a), by B56-containing PP2As and Akt. Molecular Biology of the Cell, 26(6), 1160-1173.

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Analyzing T Cell Death Pathways

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T cell death was analyzed by flow cytometric staining for Annexin V (BD Bioscience) and 7-AAD (BD Bioscience) in different conditions. For in vitro activation, cells from the spleen or LNs were activated either with anti-CD3/CD28 signaling or with PMA/ionomycin stimulation. For treatment with Z-VAD-FMK (R&D Systems), BHA (Sigma), or okadaic acid (sigma), cells from LNs were pretreated with indicated concentrations of Z-VAD-ZMK, BHA or okadaic acid for 30min, then stimulated with PMA(10ng)/Ionomycin (1000ng) for 6h. For assessment of cell death in vivo, cells in the spleen or tumors from WT and KO mice were directly stained for Annexin V and 7-AAD without any in vitro stimulation.

Rao E., Zhang Y., Zhu G., Hao J., Persson X.M., Egilmez N.K., Suttles J, & Li B. (2015). Deficiency of AMPK in CD8+ T cells suppresses their anti-tumor function by inducing protein phosphatase-mediated cell death. Oncotarget, 6(10), 7944-7958.

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Synthesis and Evaluation of SAB298 Compound

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SAB298 was synthesized in six steps according to the procedures outlined in WO 2018/049127 patent application (examples 3, 15, 17, 44, 45 and 46). Its activity was compared to UM-164 (Sigma-Aldrich, St. Louis, MO), dasatinib (BMS 354825), bosutinib (SKI-606), saracatinib, SU6656, sapitinib (AZD8931), ralimetinib (LY2228820), dacomitinib, lifirafenib (BGB-283) and SCH772984 (all from Selleckchem, Houston, TX), okadaic acid (MilliporeSigma, St. Louis, MO), trametinib (LC Laboratories, Woburn), and imatinib (NCI).

Halaban R., Bacchiocchi A., Straub R., Cao J., Sznol M., Narayan D., Allam A., Krauthammer M, & Mansour T.S. (2019). A novel anti-melanoma SRC-family kinase inhibitor. Oncotarget, 10(23), 2237-2251.

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Immunofluorescence and Cell Culture Protocols

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Rabbit pAb to human MxA (H-285) (ab-95926) was purchased from Abcam Inc. (Cambridge, MA, USA); Mouse mAb to the VSV nucleocapsid (N) designated 10G4 was a gift from Dr. Douglas S. Lyles (Wake Forest School of Medicine, NC, USA). Rabbit mAb to glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 14C10; number 2118) was obtained from Cell Signaling (Danvers, MA, USA). Respective AlexaFluor 488- and AlexaFluor 594-tagged secondary donkey antibodies to rabbit (A-11008 and A-11012) or mouse (A-21202 and A-21203) IgG were from Invitrogen Molecular Probes (Eugene, OR, USA).
Cyclosporin A (CsA) was purchased from APExBIO (Houston, TX, USA); calcien-AM, calyculin-A, okadaic acid, and tetraethylammonium chloride (TEA) (a “nonselective” K-channel blocker [31 (link)]) were purchased from Millipore-Sigma (St. Louis, MO, USA).
Lipton’s black tea, pre-ground Colombian coffee, and Propel were purchased from ShopRite supermarket (Elmsford, NY, USA). Tea was brewed by soaking one tea bag in one cup of boiling water (8 oz) for 2 min; coffee was brewed in a drip percolator (2 tablespoons coffee grounds per one cup of water). Drinking water was used from a water fountain in the laboratory. All beverages were equilibrated at 37 °C before use in cell culture experiments.

Sehgal P.B., Yuan H., Centone A, & DiSenso-Browne S.V. (2024). Oral Antiviral Defense: Saliva- and Beverage-like Hypotonicity Dynamically Regulate Formation of Membraneless Biomolecular Condensates of Antiviral Human MxA in Oral Epithelial Cells. Cells, 13(7), 590.

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Curcumin Characterization and Applications

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The chemical content of turmeric-derived curcumin (#218580100, Fisher
Scientific) was verified using standard methods (see below), with assayed
content 96.5% curcuminoids by weight, comprised of 80.6% curcumin with lesser
amounts of demethoxycurcumin [13.5%] and bisdemethoxycurcumin [2.4%])) [35 (link),36 (link)], and stock solutions prepared in DMSO. A non-oxidizable curcumin
analog, diacetyl curcumin (DAC), was synthesized as previously described [34 (link),37 (link)]. Cells were stimulated, as indicated, with recombinant human
TGFβ1 (#240-B, R&D Systems). N-acetylcysteine (NAC, #A1540914) and
nystatin (#J6048606) were purchased from Alfa Aesar. Cycloheximide (CHX,
#C7698), (#SML1169), okadaic acid (OA, #459620), chloroquine (CQ, #C6628), and
MG132 (#474791) were purchased from Millipore-Sigma. The MAPK inhibitors
SP600125 (#S1077) and SB202190 (#S1460) were purchased from Selleckchem.
Mini-PROTEAN TGX-PAGE gels (#4568046) were purchased from BioRad and PVDF
membranes (#IPFL0010) from Millipore.

Kunihiro A.G., Brickey J.A., Frye J.B., Cheng J.N., Luis P.B., Schneider C, & Funk J.L. (2021). Curcumin Inhibition of TGFβ signaling in bone metastatic breast cancer cells and the possible role of oxidative metabolites. The Journal of nutritional biochemistry, 99, 108842.

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Compound Library Preparation Protocol

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All the compounds, coming from our in-house MBC library,³² (link) were prepared with a stock concentration of 25 or 10 mM in DMSO. The final % of DMSO in cell culture was not higher than 0.1%. Bafilomycin A1 (Baf1) (Enzo Life Sciences – BML-1100–0100) and okadaic acid (OA) (Sigma – O9381) were also prepared in DMSO. Deferiprone (DFP) (Sigma − 379409) was dissolved in water. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (Sigma, C2920) was dissolved in ethanol.

Maestro I., Madruga E., Boya P, & Martínez A. (2023). Identification of a new structural family of SGK1 inhibitors as potential neuroprotective agents. Journal of Enzyme Inhibition and Medicinal Chemistry, 38(1), 2153841.

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Cytoskeletal Dynamics in Hantavirus Infection

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Vero E6 were infected with TULV at a MOI of 0.5 in a 6-well plate. Infected cells were harvested for immunofluorescence and q-RT PCR analysis at 30 days post infection. Prior to harvesting, the cells were treated with the following cytoskeletal inhibitors; 17 μM nocodazole (Sigma-Aldrich, St. Louis, MO, USA) for 60 min and 400 nM okadaic acid (OA) (Sigma-Aldrich) for 30, 60 or 90 min. Infected Vero E6 cells treated with OA were also recovered by removing the inhibitor and incubating overnight with fresh media before harvesting. Mock treatments were carried out using an equal volume of solvent (dimethyl sulfoxide (DMSO)/ethanol).

Davies K.A., Chadwick B., Hewson R., Fontana J., Mankouri J, & Barr J.N. (2020). The RNA Replication Site of Tula Orthohantavirus Resides within a Remodelled Golgi Network. Cells, 9(7), 1569.

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Maduramicin ammonium cytotoxicity mechanism

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Maduramicin ammonium (molecular weight = 934.16, purity > 97%, by HPLC) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution (5 mg/ml), aliquoted and stored at –20°C. Dulbecco’s modified Eagle’s medium (DMEM) and 0.05% trypsin-EDTA were obtained from Mediatech (Manassas, VA, USA). Fetal bovine serum (FBS) was from Atlanta Biologicals (Lawrenceville, GA, USA). Okadaic acid (OA) was from Sigma (Saint Louis, MO, USA). CellTiter 96® AQ_ueous one solution cell proliferation analysis kit was from Promega (Madison, WI, USA). Enhanced chemiluminescence solution was from Perkin-Elmer Life Science (Boston, MA, USA). The following antibodies were used: ERK2, JNK, phosphorylated-JNK (p-JNK) (Thr183/Tyr185), c-Jun, p-c-Jun (Ser63), p38, p-PP2A (Tyr307), demethylated-PP2A (De-PP2A), HA and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-ERK1/2 (Thr202/Tyr204) and p-p38 (Thr180/Tyr182) (Cell signaling technology, Beverly, MA, USA), PP2Ac, PP2A-A, PP2A-B and methylated-PP2A (Me-PP2A) (Upstate biotechnology, Waltham, MA, USA), PP5 (BD transduction laboratories, San Jose, CA, USA), FLAG (Sigma, Saint Louis, MO, USA), goat anti-mouse IgG-horseradish peroxidase and goat anti-rabbit IgG-horseradish peroxidase (Pierce, Rockford, IL, USA).

Chena X., Jiang S, & Huang S. (2017). Maduramicin-activated protein phosphatase 2A results in extracellular signal-regulated kinase 1/2 inhibition, leading to cytotoxicity in myocardial H9c2 cells. Toxicology letters, 284, 96-102.

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Okadaic Acid Nuclear Import Inhibition

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Okadaic acid (OA) (Sigma), a inhibitor which blocks the nuclear import of HDAC4 [26 (link),27 (link)], was prepared as a 10-μM stock in dimethyl sulfoxide (DMSO, Sigma) and added to culture medium 2 hours before CTS at the final concentration of 10 nM [27 (link),28 (link)].

Chen C., Wei X., Lv Z., Sun X., Wang S., Zhang Y., Jiao Q., Wang X., Li Y, & Wei L. (2016). Cyclic Equibiaxial Tensile Strain Alters Gene Expression of Chondrocytes via Histone Deacetylase 4 Shuttling. PLoS ONE, 11(5), e0154951.

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