Ser473 akt

Manufactured by Cell Signaling Technology

Sourced in United States

The (Ser473) Akt antibody is a specific antibody that recognizes the phosphorylated form of the Akt protein at serine 473. Akt is a key signaling protein involved in regulating cellular processes such as metabolism, cell growth, and survival.

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6 protocols using ser473 akt

Evaluating IGF-1R Signaling Pathways

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Total IGF-1R, IRS-1, p85, ERK1/2 and Akt and phosphorylated (Tyr1135/1136) IGF-1R, (Tyr458) p85, (Thr202/Tyr204) ERK1/2, (Ser473) Akt, and GAPDH antibody (rabbit) primary antibodies were obtained from Cell Signaling (Danvers, MA). Secondary goat anti-rabbit IgG antibodies (IRDye 800CW) were purchased from LI-COR Biotechnology (Lincoln, NE). AG 1024, a specific IGF-1R phosphorylation inhibitor, was purchased from Selleck Chemicals (Houston, TX). IGF-1R siRNA was obtained from Cell Signaling (Danvers, MA). D-mannitol and glucose were purchased from Sigma (St. Louis, MO). KZ-41 (Fig 1) was synthesized in Dr. Duane Miller’s laboratory and verified to be >96% pure by nuclear magnetic resonance spectroscopy [17 (link)].

He H., Weir R.L., Toutounchian J.J., Pagadala J., Steinle J.J., Baudry J., Miller D.D, & Yates C.R. (2017). The quinic acid derivative KZ-41 prevents glucose-induced caspase-3 activation in retinal endothelial cells through an IGF-1 receptor dependent mechanism. PLoS ONE, 12(8), e0180808.

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Quantitative Western Blot Analysis

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Primary antibody: Rabbit anti-mouse Glut4 antibody was purchased from Abcam (CAT#ab654, Cambridge, MA); Rabbit anti-mouse IRS1 (#CAT2390S), Ser612-IRS1 (CAT#3203S), Ser636/639-IRS1 (CAT#2388S), AKT (CAT#9272S), Thr308-AKT (CAT#13038), Ser-473- AKT (CAT#4060S) antibodies and the AMPK and ACC antibody sampler kit (CAT#9957) were obtained from Cell Signaling Technology (Danvers, MA); Rabbit anti-mouse Glut2 (CAT#sc-518022), IRS2 (CAT#sc-390761) and Actin antibodies (CAT#sc-58673) were purchased from Santa Cruz (Stockton, CA). Goat anti-rabbit IgG-HRP (CAT#sc-2004) was also obtained from Santa Cruz (Stockton, CA).

Xu H., Fu Q., Zhou Y., Xue C., Olson P., Lynch E.C., Zhang K.K., Wu C., Murano P., Zhang L, & Xie L. (2018). A long-term maternal diet intervention is necessary to avoid the obesogenic effect of maternal high-fat diet in the offspring. The Journal of nutritional biochemistry, 62, 210-220.

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Breast and Prostate Cancer Cell Lines

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The MDA-MB-231 and MDA-MB-468 breast cancer and PC-3 prostate cancer cell lines were purchased from American Type Culture Collection (Manassas, VA). Media used for the maintenance of these cells were as follows: MDA-MB-231 and MDA-MB-468, DMEM; PC-3, RPMI 1640, all of which were supplemented with 10% fetal bovine serum. Cells were incubated at 37°C in a humidified incubator containing 5% CO₂. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] was obtained from TCI America (Portland, OR), and the ECL Western Blotting System from GE Healthcare Life Sciences (Pittsburgh, PA). Antibodies specific for the following protein targets were used: p-Thr172-AMPK, AMPK, p-Thr389-p70S6K, p70S6K, Ser473-Akt, Akt, Foxo3a, claudin-1, vimentin, snail, and β-actin (Cell Signaling Technology, Inc., Beverly, MA); E-cadherin, BD Biosciences (San Diego, CA). Alexa Fluor 555- and 488-conjugated goat anti-rabbit and anti-mouse IgG were purchased from Invitrogen (Carlsbad, CA), and anti-mouse and anti-rabbit secondary antibodies was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).

Dokla E.M., Fang C.S., Lai P.T., Kulp S.K., Serya R.A., Ismail N.S., Abouzid K.A, & Chen C.S. (2015). Development of Potent Adenosine Monophosphate-Activated Protein Kinase Activators. ChemMedChem, 10(11), 1915-1923.

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Comprehensive Signaling Pathway Profiling

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(Ser473)-Akt, (Ser235/236)-S6, (Ser32/36)-IκB, (Thr202/Tyr204)-Erk1/2, Akt, S6, Erk1/2, IκB, p65, CD79A, Mcl1 and GAPDH antibodies were purchased from Cell Signaling, Danvers, MA, USA. Anti-NOXA was obtained from Calbiochem, La Jolla, CA, USA. Bez235, Ibrutinib, PD0325901, MG132, Bortezomib, LY294002 and BKM120 were obtained from Selleckchem, Houston, TX, USA. Temsirolimus, Orlistat and cycloheximide were purchased from Sigma, Munich, Germany. MLN4924 was obtained from ChemieTek, Indianapolis, IN, USA.

Dengler M.A., Weilbacher A., Gutekunst M., Staiger A.M., Vöhringer M.C., Horn H., Ott G., Aulitzky W.E, & van der Kuip H. (2014). Discrepant NOXA (PMAIP1) transcript and NOXA protein levels: a potential Achilles' heel in mantle cell lymphoma. Cell Death & Disease, 5(1), e1013-.

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Western Blot Analysis of Cell Signaling

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WB was performed as described [49 (link)]. Antibodies used were the following: CK1α, PARP, Mcl1, Ser45 β-catenin, Ser33/37/Thr41 β-catenin, total β-catenin, Ser473 AKT, total AKT (Cell signaling Technology, MA, USA); Mdm2 (Millipore, Italy), GAPDH (Ambion, USA); α-tubulin (Sigma-Aldrich, Italy); Bak (Merck, MA, USA); Bax, p21, p53 (Becton Dickinson, Italy); Caspase 3 (Enzo Life Science, UK). Images were acquired using the Image Quant LAS 500 chemiluminescence detection system (GE Healthcare, USA). Densitometric analysis was performed with Quantity One software (Bio-Rad, Italy).

Manni S., Carrino M., Manzoni M., Gianesin K., Nunes S.C., Costacurta M., Tubi L.Q., Macaccaro P., Taiana E., Cabrelle A., Barilà G., Martines A., Zambello R., Bonaldi L., Trentin L., Neri A., Semenzato G, & Piazza F. (2017). Inactivation of CK1α in multiple myeloma empowers drug cytotoxicity by affecting AKT and β-catenin survival signaling pathways. Oncotarget, 8(9), 14604-14619.

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Quantification of Phosphorylated Proteins

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Snap-frozen skeletal muscle (quadriceps) were lysed in ice-cold buffer containing: 50 mM Hepes (pH 7.6), 50 mM NaF, 50 mM KCl, 5 mM NaPPi, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 5 mM βglycerophosphate, 1 mM sodium vanadate, 1% NP40 and protease inhibitors cocktail (Complete, Roche, Mijdrecht, The Netherlands) as previously reported [11] . Western blots were performed using phosphospecific Ser473-Akt, (#9271), Ser2481-mTOR (#2974) and Thr389-S6K (#9205) from Cell Signaling Technology. Bands were visualized by enhanced chemiluminescence and quantified using Image J (NIH, US).

van den Hoek A.M., Zondag G.C.M., Verschuren L., de Ruiter C., Attema J., de Wit E.C., Schwerk A.M.K., Guigas B., Lek S., Rietman A., Strijker R, & Kleemann R. (2019). A novel nutritional supplement prevents muscle loss and accelerates muscle mass recovery in caloric-restricted mice. Metabolism: clinical and experimental, 97.

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