Phosphorylated akt s473

Manufactured by Cell Signaling Technology

Sourced in United States

Phosphorylated AKT S473 is a cellular protein that plays a key role in various cellular processes. It is a specific antibody that recognizes the serine 473 phosphorylation site on the AKT protein, which is a critical regulator of cell growth, survival, and metabolism. This product can be used in various research applications, such as Western blotting, immunohistochemistry, and flow cytometry, to detect and quantify the levels of phosphorylated AKT in biological samples.

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Phosphorylated Akt (120 citations) Anti-phosphorylated Akt (S473) (3 citations) Phosphorylated Akt (PAkt) S473 and T308 (2 citations) Phosphorylated AKT at S473 (2 citations) Phosphorylated-Akt (p-Akt) (S473) (2 citations)

16 protocols using phosphorylated akt s473

Cell Cycle Regulation by Lunasin

Cited 1 time

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All cell culture media and supplements were from Life Technologies, except fetal bovine serum (FBS) from Atlanta Biologicals; insulin, hydrocortisone, and bovine serum albumin fraction V (BSA) from Sigma-Aldrich; Bronchial Epithelial Cell Basal Medium and Bronchial Epithelial Growth Medium SingleQuots™ from Lonza; collagen from Advanced BioMatrix; and fibronectin from BD Biosciences. Primary antibodies against phosphorylated RB (Ser608, Ser780 and Ser807/811), cyclin D1, phosphorylated cyclin D1 (Thr286), CDK4, CDK6, p21^Waf1/Cip1, p27^Kip1, GSK-3β, phosphorylated GSK-3β (Ser9), Akt, phosphorylated Akt (S473), and β-actin were from Cell Signaling Technology. Primary antibodies against p15^INK4b, p16^INK4a, p18^INK4c, p19^INK4dand RB were from Santa Cruz Biotechnology. The phytoeryhthrin (PE)-conjugated anti-BrdU antibody and 7-aminoactinomycin D (7-AAD) nucleic acid dye were from BD Biosciences. Alkaline phosphatase (AP)-conjugated AffiniPure goat anti-rabbit IgG and horseradish peroxidase (HRP)-conjugated AffiniPure (goat anti-rabbit IgG and sheep anti-mouse IgG) were from Jackson ImmunoResearch. The nucleic acid stain 4′,6-diamidino-2-phenylindole (DAPI) was from Life Technologies. Lunasin (> 99% purity) was purified from defatted soybean flour (white flake) as previously reported by Kentucky BioProcessing [18 (link)].

McConnell E.J., Devapatla B., Yaddanapudi K, & Davis K.R. (2014). The soybean-derived peptide lunasin inhibits non-small cell lung cancer cell proliferation by suppressing phosphorylation of the retinoblastoma protein. Oncotarget, 6(7), 4649-4662.

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KIT Phosphorylation Analysis in Cells

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Western blots were performed on cell lysates from ROSA^{KIT WT} cells stimulated with SCF, or from unstimulated ROSA^{KIT D816V} and ROSA^{KIT D816V-Gluc} cells, using an antibody against phosphorylated KIT Y719 (Cell Signaling Technology), in order to visualize the phosphorylation state of KIT. Western blots were also performed on lysates of mouse BM cells using antibodies against phosphorylated KIT Y719, phosphorylated STAT5 Y694, phosphorylated AKT S473 and phosphorylated ERK Y202/204 (all from Cell Signaling Technology). Proteins were visualized with horseradish peroxidase-conjugated secondary antibodes and chemoluminescent substrate (Promega). A mouse monoclonal IgG1 anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Ab (Santa Cruz Biotechnology) was used as a loading control.

Bibi S., Zhang Y., Hugonin C., Mangean M.D., He L., Wedeh G., Launay J.M., Van Rijn S., Würdinger T., Louache F, & Arock M. (2016). A new humanized in vivo model of KIT D816V+ advanced systemic mastocytosis monitored using a secreted luciferase. Oncotarget, 7(50), 82985-83000.

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Western Blotting Analysis of Signaling Pathways

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Cell lysates were produced and Western blotting was carried out as described previously.³⁴ Primary antibodies used were all from Cell Signalling Technology: phosphorylated‐AKT (S473) (#4060), AKT (#4691), phosphorylated RELA (S536) (#3033), RELA (#8242), p STAT3 (s727) (#9134), phosphorylated‐STAT3 (Y705) (#9145), STAT3 (#4904), IKKε (#2904), TBK1 (#3013), β‐actin (#8457), GAPDH (#2118). Membranes were incubated with HRP‐coupled anti‐rabbit secondary antibody (Cell Signaling Technology), in 5% milk/BSA in TBS‐0.05% Tween‐20 for 1 hour at room temperature and visualization was with ECL (BioRad) and exposure to X‐ray film.

Carr M., Mamand S., Chapman K.L., Perrior T, & Wagner S.D. (2020). IKKε and TBK1 in diffuse large B‐cell lymphoma: A possible mechanism of action of an IKKε/TBK1 inhibitor to repress NF‐κB and IL‐10 signalling. Journal of Cellular and Molecular Medicine, 24(19), 11573-11582.

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Carnosol Modulates Cell Signaling Pathways

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Carnosol (purity: > 98% by HPLC) was purchased from Sigma-Aldrich (St. Louis, MO, USA). CNBr-Sepharose 4B beads were from GE Healthcare (Piscataway, NJ, USA). Antibodies to detect total ERKs, phosphorylated ERKs (T202/Y204), total RSK, phosphorylated RSK (S380), total AKT, phosphorylated AKT (S473), phosphorylated CREB (S133), total CREB, phosphorylated CDC2 (Y15), total CDC2, cyclin A2, cyclin B1, cyclin D1, p27(kip1), phosphorylated CDK2 (T160), total CDK2, caspase 9, cleaved caspase 7 and BcL-xL were obtained from Cell Signaling Technology (Beverly, MA, USA). The antibody to detect β-actin and p21 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Active RSK2, and ATF1 (RSK2 substrate) human recombinant protein for kinase assays were purchased from SignalChem (Richmond, BC, Canada).

Wang L., Zhang Y., Liu K., Chen H., Yang R., Ma X., Kim H.G., Bode A.M., Kim D.J, & Dong Z. (2018). Carnosol suppresses patient-derived gastric tumor growth by targeting RSK2. Oncotarget, 9(76), 34200-34212.

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Autophagy Regulation Pathway Analysis

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Antibodies used in this study were BNIP3 (#Ab10433), NIX (#Ab8399) and cyp2E1 (#Ab19140) from Abcam, p62 (#H00008878-M01) from Abnova, FoxO3 (#2497), phosphorylated Akt (S473, #4060), Akt (#2966), phosphorylated 4EBP1 (Ser65, #9451), 4EBP1 (#9452) and Lamin A/C (#2032) from Cell Signaling Biotechnology, β-actin (#A5441) from Sigma-Aldrich, MnSOD (#sc-30080) from Santa Cruz Biotechnology, and horseradish peroxidase–conjugated secondary antibody from Jackson ImmunoResearch Laboratory. The rabbit polyclonal anti-LC3B antibody was generated as described previously [10] (link). Adenovirus GFP-LC3 was generated as described previously [10] (link). Chloroquine (CQ) was from Sigma-Aldrich. Ethanol was from Pharmaco, Inc. and all other chemicals were from Sigma, Invitrogen, or Calbiochem.

Ni H.M., Bhakta A., Wang S., Li Z., Manley S., Huang H., Copple B, & Ding W.X. (2014). Role of Hypoxia Inducing Factor-1β in Alcohol-Induced Autophagy, Steatosis and Liver Injury in Mice. PLoS ONE, 9(12), e115849.

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Immunoblot Analysis of ER and Signaling Proteins

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Immunoblots were prepared as previously described (29 ). The following antibodies were used: ER (1:1000, Cell Signaling D6R2W), AREG (1:1000, Proteintech 16036–1-AP), TFF1 (1:500, Santa Cruz Biotechnologies A-10), phosphorylated Erk p44/42 (1:1000, Cell Signaling 9106S), Erk (1:1000, Cell Signaling 4695S), phosphorylated Akt S473 (1:1000, Cell Signaling 9271L), Akt (1:1000, Cell Signaling 9272S), Cyclin D1 (1:1000, Cell Signaling 2922S), phosphorylated ER S118 (1:1000, Cell Signaling 2511S), phosphorylated ER S167 (1:1000, Cell Signaling #5587P), phosphorylated ER S104/106 (1:500, Cell Signaling #2517), and FGFR1 (1:1000, Santa Cruz Biotechnologies M2F12).

DiGiacomo J.W., Godet I., Trautmann-Rodriguez M, & Gilkes D.M. (2020). Extracellular matrix-bound FGF2 mediates estrogen receptor signaling and therapeutic response in breast cancer. Molecular cancer research : MCR, 19(1), 136-149.

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Biochemical Signaling Pathway Analysis

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All reagents were purchased from Thermo Fisher Scientific unless otherwise stated. 2-MeS ADP, ADP, and Apyrase (type V) were purchased from Sigma (St. Louis, MO, USA). AR-C69931MX was a gift from the Medicines Company (Parsippany, NJ, USA). AYPGKF was purchased from GenScript (Piscataway, NJ, USA). Phosphorylated Akt S473 and phosphorylated VASP S157 were purchased from Cell Signaling Technologies (Beverly, MA, USA), while the total Akt antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The total VASP antibody was purchased from Origene (Rockville, MD, USA). Odyssey blocking buffer and secondary antibodies IRDye 800CW goat anti-rabbit and IRDye 680LT goat anti-mouse were purchased from Li-Cor (Lincoln, NE, USA). Collagen and Chronolume, used for the detection of secreted ATP, were purchased from Chrono-Log Corporation (Havertown, PA, USA). GTP-bound Rap1b was assessed using a kit from Cell Signaling and cAMP production was measured using a kit from Enzo. Prostaglandin E1 was also purchased from Enzo (New York, NY, USA). AseI was purchased from New England BioLabs (Ipswitch, MA, USA).

Dangelmaier C., Mauri B., Patel A., Kunapuli S.P, & Kostyak J.C. (2022). D121 Located within the DRY Motif of P2Y12 Is Essential for P2Y12-Mediated Platelet Function. International Journal of Molecular Sciences, 23(19), 11519.

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Western Blot Analysis of Insulin Signaling

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Tissues were processed using a multisample pulverizer and were prepared in lysis buffer (catalog #9803S; Cell Signaling Technology) containing HALT inhibitor cocktail (catalog #78442; Thermo Scientific). An equivalent amount of protein was used for all samples in each gel. Membranes were incubated with primary antibodies at 4°C overnight, as follows: Rab10 (1:500; catalog #4262; Cell Signaling Technology), GLUT4 (1:25,000; catalog #2213; Cell Signaling Technology), phosphorylated AS160 T642 (1:1,000; catalog #8881; Cell Signaling Technology), AS160 (1:5,000; catalog #07–741; Millipore), Rab8a (1:1,000; catalog #610844; BD Biosciences), phosphorylated Akt S473 (1:2,000; catalog #9271; Cell Signaling Technology), and Akt (1:1,000; catalog #9272; Cell Signaling Technology).

Vazirani R.P., Verma A., Sadacca L.A., Buckman M.S., Picatoste B., Beg M., Torsitano C., Bruno J.H., Patel R.T., Simonyte K., Camporez J.P., Moreira G., Falcone D.J., Accili D., Elemento O., Shulman G.I., Kahn B.B, & McGraw T.E. (2016). Disruption of Adipose Rab10-Dependent Insulin Signaling Causes Hepatic Insulin Resistance. Diabetes, 65(6), 1577-1589.

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Quantitative Western Blot Analysis in Metabolic Tissues

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Mouse liver, white adipose and gastrocnemius skeletal muscle was lysed in homogenization buffer. Protein concentrations were determined using the Bradford assay (Thermo Fisher) before separation by SDS-PAGE. Proteins were transferred to nitrocellulose membranes (Licor) and blocked for 10 min in blocking buffer (Licor) diluted in TBST. Membranes were then incubated with Revert stain (Licor) per manufacturer’s instructions to quantitate total protein levels. Membranes were then incubated overnight with primary antibodies followed by a 60 min incubation at room temperature with fluorescently conjugated secondary antibodies (Licor). The following primary antibodies from Cell Signaling Technologies were used at a 1:1000 dilution: phosphorylated AKT (S473), phosphorylated GSK3β (S9) and GSK3β. The following primary antibodies from ProteinTech were used at a 1:1000 dilution: AKT, SOD1, SOD2 and catalase. Fluorescent intensity was quantified using Image Studio (Licor).

Carter C.S., Huang S.C., Searby C.C., Cassaidy B., Miller M.J., Grzesik W.J., Piorczynski T.B., Pak T.K., Walsh S.A., Acevedo M., Zhang Q., Mapuskar K.A., Milne G.L., Hinton AO J.r., Guo D.F., Weiss R., Bradberry K., Taylor E.B., Rauckhorst A.J., Dick D.W., Akurathi V., Falls-Hubert K.C., Wagner B.A., Carter W.A., Wang K., Norris A.W., Rahmouni K., Buettner G.R., Hansen J.M., Spitz D.R., Abel E.D, & Sheffield V.C. (2020). Exposure to static magnetic and electric fields treats type 2 diabetes. Cell metabolism, 32(4), 561-574.e7.

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Measurement of AKT Phosphorylation and PI3K Isoforms

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For measurement of AKT phosphorylation, whole-cell lysates (20 µg) were separated by 10% SDS-PAGE and electrotransferred to polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences, Piscataway, NJ). The membranes were pre-incubated for 1 h at room temperature in Tris-buffered saline (pH 7.6) that contained 0.1% Tween-20 and 5% not-fat dry milk. The PVDF membranes were then incubated with specific antibodies against phosphorylated AKT (S473) or total AKT (Cell Signaling Technology). Total AKT was measured by stripping and reprobing. Immunoreactive bands were detected by incubating with horseradish peroxidase-conjugated anti-rabbit IgG (Cell Signaling Technology) and the bands were visualized using the ECL system (Amersham Biosciences). For measurement of p110 isoform expression, purified B, T and NK cells from C57BL/6 mice along with the SKOV and U87MG cell lines (ATCC, Manassas, VA) were lysed and 30 µg protein were separated by SDS-PAGE. Proteins were transferred to nitrocellulose and probed with the following antibodies to the different PI3K p110 isoforms: p110α (C73F8), p110β (C33D4), p110γ (D55D5) (Cell Signaling Technology), p110δ(Abcam, Cambridge, MA) and Actin (Sigma Aldrich).

Yea S.S., So L., Mallya S., Lee J., Rajasekaran K., Malarkannan S, & Fruman D.A. (2014). Effects of Novel Isoform-Selective Phosphoinositide 3-Kinase Inhibitors on Natural Killer Cell Function. PLoS ONE, 9(6), e99486.

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