Akt antibody

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom

The Akt antibody is a laboratory reagent used to detect and study the Akt protein, a key regulator of cellular processes such as metabolism, proliferation, and survival. The antibody specifically recognizes and binds to the Akt protein, allowing researchers to identify and quantify its presence in biological samples.

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Lab products found in correlation

Close spelling variants of this product

Anti-Akt antibody (116 citations) Anti-p-AKT antibody (42 citations) P-AKT antibody (30 citations) Rabbit anti-Akt antibody (23 citations) Rabbit polyclonal anti-Akt antibody (16 citations) Anti-P-Akt (Ser473) antibody (13 citations) Total Akt antibody (13 citations) Anti-AKT polyclonal antibody (9 citations) Anti-Akt monoclonal antibodies (5 citations) Anti-phospho-Akt-S473 antibody (4 citations)

71 protocols using akt antibody

Cardiomyocyte Protein Expression Analysis

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Cardiomyocytes were lysed in buffer (150 mM NaCl, 20 mM Tris, pH 7.4, 1 mM EDTA, 1% Triton X-100) containing protease and phosphatase inhibitor cocktail sets (Roche, UK) and harvested as previously described.
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Equal amounts of protein (15 µg/sample) were resolved by electrophoresis on 10% Tris-glycine gels, transferred onto Immobolin PVDF membranes (Millipore), blocked and probed with specific antibodies: Phospho-(Ser) PKC substrate, phospho-Erk1/2, (Thr202/Tyr204), Erk1/2, phospho-Akt, (Ser 473), and Akt antibodies (all 1:1,000 dilution) were from Cell Signaling Technology (Massachusetts, United States) and mouse–anti-GAPDH (1:2,000 dilution) was from Santa Cruz Biotechnology (Heidelberg, Germany). HRP-conjugated secondary antibodies (antimouse and antirabbit, both 1:10,000 dilution) were from GE Healthcare (Buckinghamshire, UK).

Walsh T.G, & Poole A.W. (2017). Platelets Protect Cardiomyocytes from Ischemic Damage. TH Open: Companion Journal to Thrombosis and Haemostasis, 1(1), e24-e32.

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Regulation of Wnt5a, Notch1, and HES1 in NHKs

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NHKs (5×10⁵) were seeded onto 60-mm plates 24 h before the treatments and cultured in media without supplements (except antibiotics) for another 24 h. They were then stimulated with 10 ng/ml of tumor necrosis factor (TNF)-α (Invitrogen), 20 ng/ml of interferon (IFN)-γ (LG Life Sciences, Iksan, Korea), 25 ng/ml of transforming growth factor (TGF)-α (Millipore, Billerica, MA, USA), and 10 ng/ml of interleukin (IL)-1α (R&D Systems, Rochester, MN, USA) and a mixture of four cytokines for 24 h. Total proteins (20 µg) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and probed with anti-Wnt5a (1:500; Imgenex, San Diego, CA, USA), -Notch1 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and -HES1 (1:500; Millipore) antibodies. β-actin (Sigma-Aldrich, St. Louis, MO, USA) was used as a loading control.
For analyses of phosphorylated proteins, NHKs were stimulated with 100 ng/ml of recombinant mouse Wnt5a (rWnt5a) (Millipore) for 15 min, 30 min, 1 h, and 3 h. Total proteins (30 µg) were resolved by SDS PAGE and probed with anti-phospho-AKT (Ser473) (Cell Signaling, 1:500) and -AKT antibodies (Cell Signaling, 1:500). Densitometric analyses were performed using a VersaDoc Imaging System (Bio-Rad, Hercules, CA, USA).

Kim J.E., Bang S.H., Choi J.H., Kim C.D., Won C.H., Lee M.W, & Chang S.E. (2016). Interaction of Wnt5a with Notch1 is Critical for the Pathogenesis of Psoriasis. Annals of Dermatology, 28(1), 45-54.

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Inflammatory Signaling Pathway Analysis

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Deoxycholic acid (DCA), lipopolysaccharide (LPS), CA-074 Me, and JTE-013 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dextran sulfate sodium (DSS, molecular weight 36–50 kDa) was obtained from MP Biomedicals Inc. (Irvine, CA, USA). ASC antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). IL-1β, ERK, AKT antibodies, and U0126 were purchased from Cell Signaling Technologies (Beverly, MA, USA). DMEM and RPMI 1640 were obtained from Invitrogen (Carlsbad, CA, USA). ELISA Kits were from eBioscience (San Diego, CA, USA).

Zhao S., Gong Z., Du X., Tian C., Wang L., Zhou J., Xu C., Chen Y., Cai W, & Wu J. (2018). Deoxycholic Acid-Mediated Sphingosine-1-Phosphate Receptor 2 Signaling Exacerbates DSS-Induced Colitis through Promoting Cathepsin B Release. Journal of Immunology Research, 2018, 2481418.

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Molecular Mechanisms of Oxidative Stress

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The compounds 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and ZnPP were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Calbiochem (San Diego, CA, USA) provided U0126 and LY294002. Santa Cruz Biotechnology (Santa Cruz, CA, USA) supplied primary HO-1, β-actin, Nrf2, ERK, and phospho-ERK antibodies. Cell Signaling Technology (Beverly, MA, USA) supplied primary phospho-Akt (Ser 473) and Akt antibodies. Epitomics Inc. (Burlingame, CA, USA) provide the secondary rabbit-specific antibody. Other reagents were purchased as analytical grade.

Ryu Y.S., Fernando P.D., Kang K.A., Piao M.J., Zhen A.X., Kang H.K., Koh Y.S, & Hyun J.W. (2019). Marine Compound 3-Bromo-4,5-dihydroxybenzaldehyde Protects Skin Cells against Oxidative Damage via the Nrf2/HO-1 Pathway. Marine Drugs, 17(4), 234.

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Aβ-induced Signaling Pathway Inhibition

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Aβ was purchased from AnaSpec (CA, USA). Anti-phospho-phosphatidylinositol-3-kinase (pPI3K), PI3K, phosphor-Akt (pAkt), and Akt antibodies were obtained from Cell Signaling Technology (MA, USA). β-amyrin and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). LY294002 and U0126 were obtained from Tocris Bioscience (MO, USA).

Park H.J., Kwon H., Lee J.H., Cho E., Lee Y.C., Moon M., Jun M., Kim D.H, & Jung J.W. (2019). β-Amyrin Ameliorates Alzheimer’s Disease-Like Aberrant Synaptic Plasticity in the Mouse Hippocampus. Biomolecules & Therapeutics, 28(1), 74-82.

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Western Blot Analysis of Signaling Pathways

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Cells were seeded in 10 cm dishes 24 hours prior to treatment and incubated with indicated compounds for 1 hour. Cells were washed with phosphate-buffered saline (PBS) and lysed with NP-40 lysis buffer (1% NP40, 150 mM NaCl, and 25 mM Tris, pH 8.0) supplemented with protease inhibitors (Complete Protease Inhibitor Cocktail, Roche) and phosphatase inhibitors (PhosSTOP, Roche). Concentration of protein was determined using Lowry assays (Bio-Rad, Hercules, CA) and equal amount of whole cell protein lysate was loaded in each lane and resolved using 4-12% gradient Bis-Tris gel (Invitrogen, CA). Proteins were transferred to 0.2-0.45μm nitrocellulose membrane (Invitrogen, CA). Membranes were incubated overnight at 4 °C with primary antibodies after blocking, followed by incubation with appropriate HRP-conjugated secondary antibody at room temperature for one hour. ECL-Plus was used to detect the activity of peroxidase according to the manufacturer's protocol (Amersham Pharmacia, Uppsala, Sweden). Antibodies raised against phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), pAKT(S473), phospho-p70 S6K and total ERK, AKT antibodies were purchased from Cell Signaling Technology (Beverly MA, USA), and anti-beta actin (conjugated with HRP) was purchased from Abcam (Cambridge, MA, USA). Secondary HRP antibodies were purchased from Jackson ImmunoResearch (St. Louis MO, USA).

Van Dort M.E., Galbán S., Wang H., Sebolt-Leopold J., Whitehead C., Hong H., Rehemtulla A, & Ross B.D. (2015). Dual Inhibition of Allosteric Mitogen-Activated Protein Kinase (MEK) and Phosphatidylinositol 3-Kinase (PI3K) Oncogenic Targets with a Bifunctional Inhibitor. Bioorganic & medicinal chemistry, 23(7), 1386-1394.

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IGF-I Signaling Pathway Analysis

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IGF-I was obtained from R&D System, Inc. (Minneapolis, MN, USA). VER-155008 and YM-08 were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Phospho-specific p44/p42 MAP kinase, p44/p42 MAP kinase, phospho-specific Akt (Thr308), and Akt antibodies were used for the first antibodies (Cell Signaling, Beverly, MA, USA). An ECL Western blotting detection kit was used (GE Healthcare UK Ltd., Buckinghamshire, UK). Other materials were purchased from commercial sources. VER-155008 and YM-08 were dissolved in dimethyl sulfoxide (DMSO). The maximum concentration of DMSO was 0.1%, which did not affect the assay for cell migration and the detection of the protein level using Western blotting.

Kawabata T., Tokuda H., Sakai G., Fujita K., Matsushima-Nishiwaki R., Kuroyanagi G., Otsuka T, & Kozawa O. (2018). HSP70 Inhibitor Suppresses IGF-I-Stimulated Migration of Osteoblasts through p44/p42 MAP Kinase. Biomedicines, 6(4), 109.

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Melittin and Protein Signaling Regulation

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Melittin was purchased from Baichun Anhui co., Ltd, batch number: 20061216 (Anhui, china). Antibodies against cyclinD1, CDK4 were purchased from Boster (Wuhan, China) or β-actin was purchased from Santa Cruz Biotechnology (California, USA), Akt antibodies, phospho-Akt antibodies, H3 antibodies and ac-H3 antibodies were purchased from Cell Signaling (Beverly, MA, USA). HDAC2 antibody was purchased from anbo (San Francisco, CA, USA). HDAC2, cyclinD1, CDK4 and PTEN primers were produced from Shanghai Sangon Biological and Technological Company (Shanghai, China). Secondary antibodies for goat anti-rabbit immunoglobulin IgG horse radish peroxidase (HRP), and goat anti-mouse IgG HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA).

Zhang H., Zhao B., Huang C., Meng X.M., Bian E.B, & Li J. (2014). Melittin Restores PTEN Expression by Down-Regulating HDAC2 in Human Hepatocelluar Carcinoma HepG2 Cells. PLoS ONE, 9(5), e95520.

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Amyloid Beta Peptide Protocol

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Aβ peptide (Human, 1–40) (Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val trifluoroacetate form), was purchased from the Peptide Institute, Inc. (Osaka, Japan). TRAP (H-Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-OH trifluoroacetate salt) was purchased from Bachem Holding AG (Budendorf, Switzerland). Phospho-specific Akt (Thr-308) antibodies and Akt antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Phospho-specific HSP27 (Ser-78) antibodies and the phosphorylated HSP27 (Ser-78) ELISA kit were purchased from Enzo Life Science, Inc. (Plymouth Meeting, PA, USA). HSP27 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The PDGF-AB ELISA kit was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). The other materials and chemicals were obtained from commercial sources. Aβ peptide was dissolved in dimethyl sulfoxide. The maximum concentration of dimethyl sulfoxide was 0.35%, which had no influence on platelet aggregation, protein detection by a Western blot analysis, or ELISA for PDGF-AB and phosphorylated HSP27.

Hori T., Mizutani D., Onuma T., Okada Y., Kojima K., Doi T., Enomoto Y., Iida H., Ogura S., Sakurai T., Iwama T., Kozawa O, & Tokuda H. (2022). Relationship between the Responsiveness of Amyloid β Protein to Platelet Activation by TRAP Stimulation and Brain Atrophy in Patients with Diabetes Mellitus. International Journal of Molecular Sciences, 23(22), 14100.

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Western Blot Analysis of Signaling Pathways

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The treated cells were detached with trypsin, centrifuged and resuspended in lysis buffer, then added to the homogenized samples with protease inhibitor, PMSF and calcineurin inhibitors. Following 30 minutes of incubation at 4°C, the samples were centrifuged at 12,000g for 15min, after which the supernatants were collected and boiled with standard SDS sample buffer, then separated by 6-10% SDS gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk at room temperature for 2h, and respectively incubated with FGFR1 antibodies, TLR4 antibodies, Akt antibodies (polyclonal rabbit anti-human, 1:1000, Cell Signaling Technology Co., USA), Phospho-Akt antibodies (polyclonal rabbit anti-human, 1:1000, Cell Signaling Technology Co., USA) and GAPDH antibodies (polyclonal rabbit anti-human, 1:1000, Cell Signaling Technology Co., USA) at 4°C overnight. After which the membranes were washed and incubated for 1h in the secondary antibody goat anti-rabbit antibodies (1:500, Cell Signaling Technology Co., Ltd, MA, USA). The immunoreactive bands were detected using the ECL detection system (Wejia Technology Co, China) according to the manufacturer's instructions.

Zhang R., Dong Y., Sun M., Wang Y., Cai C., Zeng Y., Wu Y, & Zhao Q. (2019). Tumor-associated inflammatory microenvironment in non-small cell lung cancer: correlation with FGFR1 and TLR4 expression via PI3K/Akt pathway. Journal of Cancer, 10(4), 1004-1012.

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