Phospho akt thr308

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Phospho-Akt (Thr308) is a laboratory product that detects the phosphorylation of the Akt protein at the threonine 308 residue. Akt is a key regulator of cellular processes, and its phosphorylation at this site is an important indicator of its activation status.

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Lab products found in correlation

β actin, by Santa Cruz Biotechnology (3 mentions) β catenin, by Santa Cruz Biotechnology (2 mentions) Phospho akt ser473, by Santa Cruz Biotechnology (2 mentions) Phosphoserine, by Abcam (1 mentions) Phospho enos ser1177, by Cell Signaling Technology (1 mentions) Foxo1, by Santa Cruz Biotechnology (1 mentions) Anti immunoglobulin g horseradish peroxidase conjugate, by Bio-Rad (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Anti gapdh antibody, by Bioworld Technology (1 mentions) Antibodies against phospho akt ser473, by Cell Signaling Technology (1 mentions) Biospectrum 600 imaging system, by Analytik Jena (1 mentions) Mmp2 and mmp9 antibodies, by Abcam (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Enhanced chemi luminescence (ecl), by Merck Group (1 mentions) Cyclin a, by Santa Cruz Biotechnology (1 mentions) Mln4924, by Active Motif (1 mentions) Cyclin b, by Santa Cruz Biotechnology (1 mentions) Cyclin d, by Santa Cruz Biotechnology (1 mentions) Caspase 8, by Cell Signaling Technology (1 mentions) Caspase 3, by Cell Signaling Technology (1 mentions)

6 protocols using phospho akt thr308

Protein Expression of GPBAR1 and CSE in LSEC

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To investigate protein expression of GPBAR1 and CSE, LSEC were serum starved overnight and then stimulated with 10 μM BAR501 for 18 h. In another experimental setting serum starved LSEC were incubated 5, 15, 30 and 60 minutes with 10 μM BAR501. Total lysates were prepared by solubilization of endothelial cells in E1A lysis buffer containing protease and phosphatase inhibitors. The proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad) and probed with primary antibodies CSE (Santa Cruz), GPBAR1/TGR5 (Abcam), tubulin (Sigma), phospho-Akt (Thr308—Santa Cruz), Akt (Santa Cruz), phospho-Serine (Abcam), phosphoeNOS (ser1177 –Cell Signaling), eNOS (Cell Signaling), phosphoFOXO1 (Thr24 –Santa Cruz) and FOXO1 (Santa Cruz). nitrocellulose membranes from immunoprecipitation (IP) experiments were first probed with a phospho-Serine antibody, stripped and then re-probed with the CSE antibody. Similarly, nitrocellulose membranes from IP experiments were first probed with the phospho-Akt antibody, stripped and then re-probed with the Akt antibody. The anti-immunoglobulin G horseradish peroxidase conjugate (Bio-Rad) was used as the secondary antibody, and specific protein bands were visualized using Super Signal West Dura (Pierce), following the manufacturer’s suggested protocol.

Renga B., Cipriani S., Carino A., Simonetti M., Zampella A, & Fiorucci S. (2015). Reversal of Endothelial Dysfunction by GPBAR1 Agonism in Portal Hypertension Involves a AKT/FOXOA1 Dependent Regulation of H2S Generation and Endothelin-1. PLoS ONE, 10(11), e0141082.

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Western Blotting Analysis of Protein Expressions

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Western blotting was conducted after separation of polypeptides using SDS-PAGE. Proteins on gel were transferred to PVDF membrane (Merck Millipore; Billerica, MA, USA). The membrane was further incubated with indicated primary and appropriate secondary antibodies. Antibodies against ACOX1 were obtained from Proteintech Group (Chicago, IL, USA). Phosphorylated form of ERK1/2 and ERK1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and BD Biosciences, respectively. Antibodies targeting MMP3, MMP10 and phospho-Akt (Thr308) were also purchased from Santa Cruz Biotechnology. Antibodies against phospho-Akt (Ser473) were obtained from Cell Signaling Technology (Danvers, MA, USA). MMP2 and MMP9 antibodies were purchased from Abcam (Cambridge, Cambridgeshire, UK). An anti-GAPDH antibody (BioWorld; St. Louis Park, MN, USA) was used as control. HRP-conjugated secondary antibodies against rabbit IgG or mouse IgG (GeneTex) were incubated with membrane for 1 h at room temperature. Immunobands were detected using chemiluminescent HRP substrates (ECL; Merck Millipore) and captured by UVP BioSpectrum 600 Imaging System. The intensity of bands was quantified by Image J software.

Lai Y.H., Liu H., Chiang W.F., Chen T.W., Chu L.J., Yu J.S., Chen S.J., Chen H.C, & Tan B.C. (2018). MiR-31-5p-ACOX1 Axis Enhances Tumorigenic Fitness in Oral Squamous Cell Carcinoma Via the Promigratory Prostaglandin E2. Theranostics, 8(2), 486-504.

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Protein Expression and Kinase Assays

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Lithium chloride (LiCl) and d-(+)-glucose were obtained from Sigma-Aldrich, whereas Go6976 and MG132 were purchased from Merck Millipore (Darmstadt, Germany). MLN-4924 was purchased from Active Biochem (Hong Kong, China). An anti-FLAG monoclonal antibody was purchased from Sigma-Aldrich. Anti-hemagglutinin (HA) polyclonal antibody, phospho-Rb (Ser-807/811), Rb, CDK2, CDK4, cyclin A, cyclin B, cyclin D, cyclin E, p21, p27, phospho-GSK3β (Ser-9), GSK3β, phospho-AMP-activated protein kinase (Thr-172), AMP-activated protein kinase, OGT, OGA, phospho-Akt (Thr-308), Akt, β-actin, β-catenin, and TCF4 antibodies were obtained from Santa Cruz Biotechnology, Inc. Antibodies for cytochrome c, caspase-3, caspase-8, caspase-9, phospho-mTOR (Ser-2481), phospho-mTOR (Ser-2448), mTOR, phospho-P70S6K (Thr-389), P70S6K, LC3, phospho-ERK1/2 (Thr-202/Tyr-204), and ERK were purchased from Cell Signaling Technology (Danvers, MA). Mono- and polyubiquitinated antibodies were obtained from Enzo Life Sciences (Farmingdale, NY). The horseradish peroxidase-conjugated secondary antibodies were from Bethyl Laboratories (Montgomery, AL).

Choi S.W., Song J.K., Yim Y.S., Yun H.G, & Chun K.H. (2015). Glucose Deprivation Triggers Protein Kinase C-dependent β-Catenin Proteasomal Degradation. The Journal of Biological Chemistry, 290(15), 9863-9873.

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Regulation of hUCB-MSCs by Signaling Pathways

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hUCB-MSCs were obtained from MEDIPOST Co. Ltd (Seoul, Korea). Fetal bovine serum was bought from BioWhittaker Inc. (Walkersville, MO, USA). AA, A23187, bisindolylmaleimide I, BrdU, Indomethacin, LY294002, mitomycin C, Nordihydroguaiaretic acid (NDGA), rapamycin, 1-Aminobenzotriazole (1-ABT), and SB203580 were aquired from the Sigma Chemical Company (St Louis, MO, USA). Phospho-Akt^ser473, phospho-Akt^thr308, Akt1/2/3, β-Actin, collagen1A, collagen3A, collagen5A, fibronectin, phospho-p38, p38, phospho-JNK, JNK, p-ERK1/2, ERK, lamin A/C, MMP-12, pan-cadherin, PKCα, PKCɛ, PKCθ, PKCζ, PKC, Phospho-PKCζ, and Sp1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-PKC, phospho-mTOR^ser2481 (mTORC2), phospho-mTOR^ser2448 (mTORC1), and mTOR were purchased from Cell Signaling (Beverly, MA, USA). The Akt inhibitor I was aquired from Calbiochem (La Jolla, CA, USA). The CD34, GPR40, phospho-Sp1, and MT3-MMP antibodies were obtained from Abcam (Cambridge, MA, USA). Mithramycin A was purchased from Tocris (Bristol, UK). Zymogram gels were bought from Novex (San Diego, CA, USA). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit IgG were obtained from Jackson Immunoresearch (West Grove, PA, USA). All other reagents were used as received and were of the highest purity commercially available.

Oh S.Y., Lee S.J., Jung Y.H., Lee H.J, & Han H.J. (2015). Arachidonic acid promotes skin wound healing through induction of human MSC migration by MT3-MMP-mediated fibronectin degradation. Cell Death & Disease, 6(5), e1750-.

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Protein Expression Analysis in Stem Cells

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Appropriate cells were lysed in RIPA lysis buffer containing a cocktail of protease inhibitors (Roche) and PMSF. Total protein concentration was determined using the bicinchoninic acid (BCA) assay kit (Ding Guo Biotechnology, Cat: BCA02). Antibodies against the following proteins were used for immunoblotting: NANOG (CST, Cat: 4893), ALDH (BD Pharmingen, Cat: 611195), OCT4 (CST, Cat: 2750), CD133 (CST, Cat: 64326), SOX2 (CST, Cat: 2748), phospho-AKT (Thr308) (CST, Cat: 2965), AKT (CST, Cat: 4691), phospho-GSK-3β (Ser9) (CST, Cat: 9323), GSK-3β (CST, Cat: 9315), β-catenin (CST, Cat: 8480, and β-actin (Santa Cruz Biotechnology, Cat: sc-47778). The immunoblots were scanned using an Odyssey infrared imaging system (LI-COR). Immunolabeling was detected using the ECL reagent (Sigma). Protein expression was normalized against β-actin.

Gan G., Shi Z., Liu D., Zhang S., Zhu H., Wang Y, & Mi J. (2021). 3-hydroxyanthranic acid increases the sensitivity of hepatocellular carcinoma to sorafenib by decreasing tumor cell stemness. Cell Death Discovery, 7, 173.

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Protein Purification and AKT Signaling

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Protein was purified from cells in culture and samples using the Thermo Scientific protein purification kit. Western Blotting was performed using antibodies against AKT (C-20, Santa Cruz Biotechnology), Phospho-AKT Ser-473 (Santa Cruz Biotechnology), Phospho-AKT Thr-308 (Santa Cruz Biotechnology), Phospho-FoxO1 (Thr24)/FoxO3A (Thr32) (Cell Signaling), PP2A A (Upstate), PP2A C (1D6, Upstate; polyclonal, Life Technologies), PP2A B55α (Upstate), PP2A B56γ [53 (link)], SET (H-120, Santa Cruz Biotechnology), and vinculin (VIN-11-5, Sigma). Immunoprecipitation was performed using protein A agarose and incubating with either AKT antibody or IgG as a negative control. Microcystin binding assay was performed using microcystin agarose beads (Upstate). For a negative control, lysates were incubated with 1.0 μM okadaic acid, a potent inhibitor and binder of the PP2A C subunit that interferes with binding to the microcystin beads.

Shouse G., de Necochea-Campion R., Mirshahidi S., Liu X, & Chen C.S. (2016). Novel B55α-PP2A mutations in AML promote AKT T308 phosphorylation and sensitivity to AKT inhibitor-induced growth arrest. Oncotarget, 7(38), 61081-61092.

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