Anti phospho akt s473

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-phospho-Akt (S473) is a primary antibody that specifically recognizes the serine 473 phosphorylated form of the Akt/PKB protein. Akt is a key regulator of various cellular processes, including cell growth, proliferation, and survival.

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Anti‐phospho‐AKT (S473) (D9E) XP™ (2 citations) Anti-AKT and anti-phospho-AKT S473 (2 citations) Anti-phospho- Akt (S473) and anti-Akt (pan, (2 citations) Anti-phospho-Akt (S473) rabbit antibody (2 citations) Anti-phospho-Akt-S473 antibody (4 citations) Phospho-AKT S473 (178 citations) Anti-phospho-Akt (493 citations) Phospho-Akt (892 citations) Anti-AKT (1447 citations)

67 protocols using anti phospho akt s473

Immune Profiling and Cytotoxicity Assay

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Anti-CD47-APC, anti-CD47-FITC, anti-CD11c-APC-Vio770, anti-CD11-PE, anti-CD14-FITC, anti-CD80-PE, anti CD83-PE, anti-CD86-PE, anti-HLA-DR-FITC, anti-HLA-ABC-FITC, anti-Hsp70-FITC, and 7-Amino-Actinomycin D (7-AAD) fluorescent DNA dye were purchased from Miltenyi. All monoclonal antibodies (mAbs) used in flow cytometry experiments were used at 1:200 titer unless otherwise specified. Anti-calreticulin-FITC mAb (EPR3924; 1:50) and anti-GAPDH were from Abcam. Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell. Anti-phospho-EGFR (Y1608), anti-EGFR, and anti-phospho-Akt (S473) were from Cell Signaling Technology. Secondary antibodies anti-rabbit IgG-HRP or anti-mouse IgG+IgM+IgA-HRP were from Bethyl Laboratories. Gefitinib was purchased from Selleckchem, and its IC₅₀ was determined for each cell line using Cell-Counting Kt-8 (Dojindo Laboratories) according to the manufacturer's instructions. Gefitinib IC₅₀ for each cell line, reported in Table 1, was used for all the experiments. Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) was purchased from Miltenyi, and interferon (IFN)-α (IntronA) from SP Europe.

Nigro A., Ricciardi L., Salvato I., Sabbatino F., Vitale M., Crescenzi M.A., Montico B., Triggiani M., Pepe S., Stellato C., Casolaro V, & Dal Col J. (2020). Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC. Frontiers in Immunology, 10, 3135.

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Western Blot Analysis of Liver Proteins

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For the Western blot analysis, the liver tissues or primary hepatocytes were lysed in 1× radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktails (Roche Diagnostic GmbH, Mannheim, Germany). Protein concentration was determined using a Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA, USA). The cell lysate was loaded onto a 10% Sodium dodecyl sulfate (SDS)-polyacrylamide gel for electrophoresis and then electrotransferred onto polyvinylidene difluoride membranes. The membranes were incubated with the indicated primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence solution (NEN Life Science, Boston, MA, USA). The following antibodies were used for the experiments: Anti-GNMT (catalog number: 14-1, YMAC Bio Tech, Taipei, Taiwan), anti-Akt (catalog number: #4685), anti-phospho-Akt T308 (catalog number: #13038), anti-phosphoAkt S473 (catalog number: #9271), anti-ERK1/2 (catalog number: #4695), anti-phosphoERK1/2 (catalog number: #4376) (Cell Signaling Technology, Danvers, MA, USA), anti-Angptl8 (catalog number: TA326696) (Origene, Rockville, MD, USA), and anti-Actin (Sigma-Aldrich) antibodies.

Huang J.W., Chen C.J., Yen C.H., Chen Y.M, & Liu Y.P. (2019). Loss of Glycine N-Methyltransferase Associates with Angiopoietin-Like Protein 8 Expression in High Fat-Diet-Fed Mice. International Journal of Molecular Sciences, 20(17), 4223.

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Western Blot and Immunostaining Antibodies

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Antibodies used for Western Blot were from: i) Cell Signaling Technology (Denver, MA), anti-phospho-Akt (S473) (#4058), anti-Akt1 (#2938), anti-Akt2 (#2964), anti-phospho-GSK3-αβ (Ser21/9) (#9331), anti-GSK3 (#9338), anti-STAT3 (#9139), anti-phospho-STAT3 (Y705) (#9145), anti-phospho-IkKα/β (Ser176/180) (#2697), anti-IkB (#9242), anti-phospho-IkB (Ser32) (#2859), anti-PTEN (#9552), anti-p110α (#4255); ii) Santa Cruz Biotechnology (Santa Cruz, CA), anti-IkKα (sc-7218); iii) Sigma-Aldrich, anti-β-actin (clone AC-74, #A2228).
Antibodies used for immunostaining were selected according to previously published work: anti-CK7 (#35057) (Menarini, Florence, Italy), anti-CK34 (Enzo Diagnostics, Inc, Farmigale, NY), anti-pAkt Ser473 (#3787) (Cell Signaling Technology), anti-pSTAT3 Tyr705 (#9145) (Cell Signaling Technology), anti-IL-6 (AF-206-NA) (R&D Systems, Minneapolis, MN).
Protein preparation and Western blot analysis were carried out by standard methods [27 (link)]. Densitometric analysis of gel bands was carried out with ImageJ software (NIH).

Malanga D., De Marco C., Guerriero I., Colelli F., Rinaldo N., Scrima M., Mirante T., De Vitis C., Zoppoli P., Ceccarelli M., Riccardi M., Ravo M., Weisz A., Federico A., Franco R., Rocco G., Mancini R., Rizzuto A., Gulletta E., Ciliberto G, & Viglietto G. (2015). The Akt1/IL-6/STAT3 pathway regulates growth of lung tumor initiating cells. Oncotarget, 6(40), 42667-42686.

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Subcellular Localization of ZDHHC22

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Cells were seeded on coverslips and then transfected with pCMV6-Entry, pCMV6-ZDHHC22, or pCMV6-ZDHHC22-(C111A). After 48 hours, cells were fixed with 4% paraformaldehyde for 20 min and then permeabilized with 0.5% Triton X-100 for 10 min. After blocking with blocking buffer, cells were incubated with primary antibodies overnight at 4°C and then incubated with secondary antibodies for 1 hour at 37°C. DAPI (Roche, Palo Alto, CA, USA) was used for DNA counterstaining. Photomicrographs were acquired with a confocal laser scanning microscope (Leica, Hilden, Germany). The following antibodies were used for immunofluorescence: anti-Flag (1:400, #14793, Cell Signaling Technology), anti-mTOR (1:200, #2983, Cell Signaling Technology), anti-phospho-AKT(S473) (1:200, #4060T, Cell Signaling Technology), anti-PHLPP2 (1:40, ab71973, Abcam), ER-Tracker Red (1:2000, C1041, Beyotime), CoraLite488 (1:200, SA00013-1, Proteintech), and CoraLite594 (1:200, SA00013-4, Proteintech).

Huang J., Li J., Tang J., Wu Y., Dai F., Yi Z., Wang Y., Li Y., Wu Y., Ren G, & Xiang T. (2022). ZDHHC22-mediated mTOR palmitoylation restrains breast cancer growth and endocrine therapy resistance. International Journal of Biological Sciences, 18(7), 2833-2850.

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Western Blot Analysis of Cellular Proteins

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We resolved cell proteins by SDS-PAGE before electro-blotting onto PVDF membranes. We incubated the membranes with the following primary antibodies overnight: anti-NCYM (1∶1000 dilution), anti-MYCN antibody (1∶1000 dilution; Calbiochem and Cell Signaling), anti-Lamin B (1∶1000 dilution; Calbiochem), anti-α-tubulin (1∶1000 dilution; Santa Cruz, CA, USA), anti-GST (1∶1000; Santa Cruz), anti-GSK3β (1∶1000 dilution; Cell Signaling), anti-phospho-GSK3β (S9) (1∶1000 dilution; Cell Signaling), anti-β-catenin (1∶1000 dilution; Cell Signaling), anti-phospho-AKT (S473) (1∶1000 dilution; Cell Signaling), anti-phospho-AKT (S308) (1∶1000 dilution; Cell Signaling), anti-AKT (1∶1000 dilution; Cell Signaling), anti-S6K (1∶1000 dilution; Cell Signaling), anti-phospho-S6K (T389) (1∶1000 dilution; Cell Signaling), and anti-actin (1∶4000 dilution; Sigma). The membranes were then incubated with a horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG at 1∶2000–1∶4000 dilution or anti-mouse IgG at 1∶2000 dilution; both from Cell Signaling Technology) and the bound proteins were visualized using a chemiluminescence-based detection kit (ECL and ECL pro kit, Amersham, Piscataway, NJ, USA; ImmunoStar LD, Wako).

Suenaga Y., Islam S.M., Alagu J., Kaneko Y., Kato M., Tanaka Y., Kawana H., Hossain S., Matsumoto D., Yamamoto M., Shoji W., Itami M., Shibata T., Nakamura Y., Ohira M., Haraguchi S., Takatori A, & Nakagawara A. (2014). NCYM, a Cis-Antisense Gene of MYCN, Encodes a De Novo Evolved Protein That Inhibits GSK3β Resulting in the Stabilization of MYCN in Human Neuroblastomas. PLoS Genetics, 10(1), e1003996.

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mTOR Modulation Impacts Endothelial Angiogenesis

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Medium 199 (M199), Hank's Balanced Salt Solution (HBSS), fetal bovine serum (FBS), and endothelial cell growth supplement were purchased from Invitrogen (Burlington, ON). VEGF-A was from Peprotech (Princeton, NJ). Rapamycin, PP242, a highly specific mTOR active-site inhibitor [12 (link)], and anti-tubulin-α was from Millipore (Temecula, CA). Ku-0063794, a second specific mTOR inhibitor [13 (link)] was from Sigma (St. Louis, MO). Anti-Akt1 was from Protein Tech (Chicago, IL). Hiperfect, non-silencing short interfering RNA (siRNA) and Akt1 silencing siRNA were from Qiagen Inc (Mississauga, ON). Human tumor necrosis factor-α (TNFα) was from Cedarlane (Mississauga, ON). Cycloheximide, phalloidin-FITC, anti-vinculin, and DAPI were from Sigma. Anti-S6K was from Abcam (Cambridge, UK). Anti-phospho-Akt^S473, anti-phospho-S6K^T389, anti-FAK, anti-phospho-FAK^Y397, anti-raptor, anti-rictor, and rictor siRNA were from Cell Signaling Technology (Danvers, MA). ON-TARGETplus human raptor siRNA-SMARTpool was from Thermo Scientific (Waltham, MA).

Farhan M.A., Carmine-Simmen K., Lewis J.D., Moore R.B, & Murray A.G. (2015). Endothelial Cell mTOR Complex-2 Regulates Sprouting Angiogenesis. PLoS ONE, 10(8), e0135245.

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Molecular Mechanisms of VEGF Signaling

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Anti-VEGFR2, anti-PLCγ, anti-HSP90, anti-Raf-1, anti-cyclin D1, anti-CDK4 and anti-p21 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-VEGFR2, anti-phospho-VEGFR2 (Y1175), anti-phospho-PLCγ (Y783), anti-phospho-Erk (T202/Y204), anti-Erk, antiphospho-Akt (S473) and anti-Akt antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-eNOS and anti-phospho-eNOS (S1177) antibodies were purchased from BD Biosciences (San Diego, CA, USA). Anti-α-tubulin antibody was purchased from Sigma-Aldrich. Recombinant human VEGF165 and 4,5-diaminofluorescein diacetate (DAF2-DA) and DAPI (4′,6-diamidino-2-phenylindole dihydrochloride hydrate) were purchased from Sigma-Aldrich. DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin) and NOC-18 was purchased from Santa Cruz Biotechnology.

Tae N., Hung T.M., Kim O., Kim N., Lee S., Na S., Min B.S, & Lee J.H. (2017). A cassaine diterpene alkaloid, 3β-acetyl-nor-erythrophlamide, suppresses VEGF-induced angiogenesis and tumor growth via inhibiting eNOS activation. Oncotarget, 8(54), 92346-92358.

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Akt and PDK1 Activation Assay

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Inactive Akt1, active Akt, active PDK1 and ATP/Mg²⁺ cocktail were purchased from Upstate Cell Signaling Solutions. Mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2) was purchased from Calbiochem. 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phospho-L-serine (18:0,22:6-PS), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (18:0, 22:6-PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0, 18:1-PC), and 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoinositol-3,4,5-trisphosphate (18:0, 20:4-PIP₃) were purchased from Avanti Polar Lipids. Sequencing grade modified trypsin was purchased from Promega. Anti-phospho-Akt (T308) (Cat#: 2965), anti-phospho-Akt (S473) (Cat#: 9271), anti-Akt (Cat#: 9272), anti-protein kinase C zeta (PKCζ)(Cat#: 9368), anti-phospho-PKCζ(T410) (Cat#: 9378), anti-phospho-PDK1(S241) (Cat#: 3061), anti-PDK1 (Cat#: 3062), and anti-GAPDH (Cat#: 2118) antibodies were purchased from Cell Signaling Technology. Anti-phospho-Akt (T34) (Cat#: 23509) was obtained from Abcam. A dilution of 1:1000 was used in western blotting for all of these antibodies. Insulin-like growth factor-1 (IGF-1) was purchased from PeproTech. PBS without Ca²⁺ was purchased from Quality Biological, Inc. Other reagents were purchased from Sigma or Quality Biological, Inc.

Huang B.X., Lee R., Akbar M, & Kim H.Y. (2015). Threonine 34 phosphorylation by phosphoinositide-dependent protein kinase 1 facilitates dissociation of Akt from the plasma membrane. The international journal of biochemistry & cell biology, 64, 195-201.

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Protein Expression and Western Blot Analysis

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Human embryonic kidney 293T cells were transfected with gene of interest for 24 h. The cells were harvested and lysed in RIPA lysis buffer (1% NP-40, 20 mM TrisCl, pH 7.5, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% sodium deoxycholate, 1 mM Na₃VO₄). Protein estimation was carried out using BCA Protein Assay Kit (Pierce, Thermo Scientific, United States). An equal amount of protein was loaded on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and was transferred to nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk (Himedia Laboratories, India). The primary antibodies used were anti-AKT, anti-Mdm2, anti-AKT substrate, anti-GAPDH, anti–phospho-AKT (S473) (Cell Signaling Technology), anti-Myc, anti-HA (Clontech), anti-GST (Santa Cruz Biotechnology), and anti-Vif (NIH, MD, United States). The secondary antibodies used were anti-rabbit/mouse–horseradish peroxidase–conjugated (Jackson ImmunoResearch). Blots were developed using ECL (enhanced chemiluminescence) reagent.

Lata S., Sood V, & Banerjea A.C. (2022). Human Immunodeficiency Virus Type 1 Vif Up-Regulates the Expression of Tat via AKT Signaling Pathway: Role of Ubiquitin Specific Protease 17. Frontiers in Microbiology, 13, 828430.

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EGFR-Mediated Signaling Pathway Analysis

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Protein was extracted from whole-cell lysates with the Mammalian Protein Extraction Reagent M-PER (Thermo Scientific Pierce) supplemented with a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich) and was quantified with the BCA Protein Assay Kit (Thermo Scientific Pierce). Lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. We performed immunoblotting using protein lysates extracted from HEK293T cells expressing empty vector, EGFR wild-type, and EGFR p.Cys326Phe plasmids. We blotted for anti-phospho-AKT S473 (Cell Signaling #4060), anti-total AKT (Cell Signaling #9272), anti-phospho-ERK1/2 (Cell Signaling #9101), anti-ERK1/2 (Cell Signaling #9102), and anti-GAPDH (Cell Signaling #2118) antibodies, at the recommended dilutions. EGFR control cell extracts (Cell Signaling #5634) were used as negative and positive controls of EGFR phosphorylation/activation. Blots were scanned digitally and quantified with the Odyssey Infrared Imaging System (Li-Cor Biosciences).

Colby S., Yehia L., Niazi F., Chen J., Ni Y., Mester J.L, & Eng C. (2016). Exome sequencing reveals germline gain-of-function EGFR mutation in an adult with Lhermitte–Duclos disease. Cold Spring Harbor Molecular Case Studies, 2(6), a001230.

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