Anti akt3

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-Akt3 is an antibody product that specifically detects Akt3, a member of the Akt serine/threonine protein kinase family. It is intended for use in Western blotting applications to identify and quantify Akt3 expression levels.

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12 protocols using anti akt3

Western Blot Analysis of Akt3 and PI3K

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Whole-cell lysates and total exosomal proteins were prepared by using RIPA buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate). 50ug total proteins were electrophoretically separated on 4–12% SDS-acrylamide Gel (Thermo Fisher Scientific). Akt3 and PI3K p110α were examined in the study. Western blot analyses were performed with primary antibodies: anti- β-actin, anti-Akt 3, anti-P-Akt, anti-PI3K, anti-VEGFA (1: 1000, Cell Signaling Technology, USA), and the corresponding secondary antibodies anti-mouse and anti-rabbit peroxidase-linked (1: 10 000; Cell Signaling Technology, USA). The signals were visualized by ECL Prime Western Blotting Detection Reagent (Advansta, USA).

Liu L., Ye Q., Liu L., Bihl J.C., Chen Y., Liu J, & Cheng Q. (2020). C6-ceramide treatment inhibits the proangiogenic activity of multiple myeloma exosomes via the miR-29b/Akt pathway. Journal of Translational Medicine, 18, 298.

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Western Blot Analysis of Protein Signaling

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After treated with TMZ for 4 days, the cells were lysed using M-PER lysis buffer. Protein was extracted and quantified using a BCA protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). A total of 30 μg of each sample was heated at 95°C for 10 min and loaded into 10% gel. Samples were electrophoresed at 110 V for 60–90 min and then transferred to PVDF membranes at 300 mA for 150 min using a semi-dry transfer apparatus. Membranes were blocked in 5% non-fat dry milk for 2 h, incubated with primary antibodies [anti-AKT3, anti-RB1, anti-MAPK8 and anti-PIK3R1 (Cell Signaling Technology, Inc., Danvers, MA, USA)] overnight, washed with TBS containing 0.05% Triton-X 100 (TBST) followed by an incubation of 1 h in goat anti-rabbit secondary antibody (1:5,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) conjugated with HRP. After final washing with TBST, the membranes were developed using chemiluminescence and exposed to X-ray film. The immunoblots were quantified with software quantity one version 4.6.2. The expression levels of AKT3, RB1, MAPK8 and PIK3R1 in each sample were internally normalized to GAPDH and levels were given relative to expression in NC group.

Wang P., Ye J.A., Hou C.X., Zhou D, & Zhan S.Q. (2016). Combination of lentivirus-mediated silencing of PPM1D and temozolomide chemotherapy eradicates malignant glioma through cell apoptosis and cell cycle arrest. Oncology Reports, 36(5), 2544-2552.

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Western Blot Analysis of Apoptosis-Related Proteins

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Cells were washed three times with cold PBS and lysed on ice with RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing a protease and phosphatase inhibitor cocktail. The concentrations of protein were determined using a BCA protein assay kit (Solarbio, Beijing, China). Equal amounts of protein were separated by SDS-PAGE and transferred electrophoretically onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked in 5% skimmed milk powder dissolved in TBST for 2 h at room temperature and subsequently incubated overnight at 4 °C with the appropriate primary antibodies: anti-AKT3 (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-cyclin D1 (1:1000, Bioss, Beijing, China), anti-CDK4 (1:1000, Bioss, Beijing, China), anti-cleaved caspase-9 (1:1000, Cell Signaling Technology), anti-cleaved caspase-3 (1:1000, Cell Signaling Technology) and anti-GAPDH (1:10,000, Absin, Shanghai, China). Then, the membranes were washed with TBST three times and incubated for 90 min with a horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature. The blots were detected with an ECL chemiluminescence solution and autoradiography with X-ray film. GAPDH was used as an internal control.

Liu H.Z., Shan T.D., Han Y, & Liu X.S. (2020). Silencing long non-coding RNA CASC9 inhibits colorectal cancer cell proliferation by acting as a competing endogenous RNA of miR-576-5p to regulate AKT3. Cell Death Discovery, 6, 115.

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Inhibition of Akt and Necroptosis Pathways

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The following materials were purchased from commercial companies: TNFα (PeroTech; Rocky Hill, NJ, USA); pan-caspase inhibitor z-VAD-fmk (Abcam, Cambridge, MA, USA). InSolution Akt Inhibitor viii isozyme-selective, Akti-1/2 and InSolution rapamycin were obtained from Calbiochem (San Diego, CA, USA). MitoSox Red was obtained from Invitrogen (Carlsbad, CA, USA). Hoechst 33258, butylated hydroxyanisole (BHA) and rotenone were obtained from Sigma (St. Louis, MO, USA). Nec-1 (5-(7-chloro-1H-indol-3-ylmethyl)-3-methylimidazolidine-2,4-dione), the inactive analog of necrostatin-1 analog (Nec-1i; 5-(7-chloro-1H-indol-3-ylmethyl)imidazolidine-2,4-dione),^{3 (link), 12 (link), 13 (link)} was a kind gift from Dr. Greg Cuny. LOX-1 was obtained from Chembridge (compound 5680672; San Diego, CA, USA), and Bai was from Cayman Chemicals (Ann Arbor, MI, USA). Antibodies were obtained from commercial sources: anti-pAkt-473, anti-p–Akt-308, anti-p-GSK-3β, anti-p-mTOR, anti-p-S6, anti-mTOR, anti-Akt1, anti-Akt2, anti-Akt3, and anti-Akt from Cell Signaling (Danvers, MA, USA); anti-RIPK1 from BD Transduction Laboratories (Lexington, KY, USA); anti-RIPK3 from ProSci Incorporated (San Diego, CA, USA); anti-HMGB1 and β-actin were obtained from Abcam.

Liu Q., Qiu J., Liang M., Golinski J., van Leyen K., Jung J.E., You Z., Lo E.H., Degterev A, & Whalen M.J. (2014). Akt and mTOR mediate programmed necrosis in neurons. Cell Death & Disease, 5(2), e1084-.

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Western Blot Analysis of Cell Signaling Proteins

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Whole-cell lysates (50 μg) were separated by 10% SDS-PAGE, and the proteins were transferred to nitrocellulose membrane. The following antibodies were used for Western blotting: anti-cyclin D1 from Neomarker (Fremont, CA); anti-Bcl-Xs from Calbiochem (Darmstadt, Germany); anti-β-Tubulin (sc-9104), anti-β-actin (sc-47778), anti-p53 (sc-6243G), anti-p57 (sc-1040), anti-p27 (sc-528), anti-Cytochrome C (SC-7159), anti-Bax (sc-7480), anti-BCL-2 (sc-7382), anti PARP (sc-7150), anti-Caspase 9 (sc-8355), anti-Caspase 3 (sc-7148) were from Santa Cruz Biotechnology (Santa Crus, CA); anti-GDI from RTG Solutions (Gaithersburg, MD); anti-Akt1 (#2967 ), anti-Akt2 ( #5239 ), anti-Akt3 (#4059 ), anti-p-y-H2A ( #9718s ) were from Cell Signaling.

Yu Z., Xu Z., DiSante G., Wright J., Wang M., Li Y., Zhao Q., Ren T., Ju X., Gutman E., Wang G., Addya S., Li T., Xiang Z., Wang C., yang X., Yang X, & Pestell R. (2014). miR-17/20 sensitization of breast cancer cells to chemotherapy-induced apoptosis requires Akt1. Oncotarget, 5(4), 1083-1090.

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Western Blotting Antibody Panel for Akt Signaling

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Anti-Akt1, anti-Akt2, anti-Akt3, anti-phospho-Akt S473 (pAkt), anti-phospho-Akt1 S473 (pAkt1), anti-phospho-Akt2 S474 (pAkt2), anti-Bax, anti-p53, anti-PUMA, anti-IGF1Rβ, anti-pGSK3β, anti-GSK3β, anti-p21, anti-PRAS40, anti-pPRAS40, anti-HA and anti-active caspase 3 antibodies were obtained from Cell Signaling Technology. Anti-β-actin antibody was purchased from Sigma-Aldrich. Anti-p85 polyclonal antibody was generated in-house and has been described (51 (link)). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit immunoglobulin G (IgG) antibody were purchased from Chemicon.

Chin Y.R., Yuan X., Balk S.P, & Toker A. (2014). PTEN-DEFICIENT TUMORS DEPEND ON AKT2 FOR MAINTENANCE AND SURVIVAL. Cancer discovery, 4(8), 942-955.

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Western Blot Analysis of Apoptosis Markers

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Cells were collected and western blot analyses were performed as previously described.^{33 (link)}β-actin was used as the loading control. The primary antibodies used for western blotting included: rabbit anti-cleaved PARP (#9544, Cell Signaling, Beverly, MA, USA), anti-H2AX (#9718, Cell Signaling), anti-Akt1 (#9514, SBA), anti-Akt2 (#8715, SBA), anti-Akt3 (#4059, Cell Signaling), anti-pAkt (#4060, Cell Signaling) and anti-β-actin (#4970, Cell Signaling).

Tan P.X., Du S.S., Ren C., Yao Q.W., Zheng R., Li R, & Yuan Y.W. (2014). MicroRNA-207 enhances radiation-induced apoptosis by directly targeting akt3 in cochlea hair cells. Cell Death & Disease, 5(10), e1433-.

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Investigating AKT Signaling Pathway Dynamics

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The following antibodies were purchased from Cell Signaling Technology: anti-phospho-AKT (Ser473) (#4060), anti-phospho-AKT (Thr308) (#13038), anti-total AKT (#9272), anti-AKT1 (#2938), anti-AKT2 (#3063), anti-AKT3 (#8081), anti-MARCKS (#5607), anti-phospho-MARCKS (Ser152/156) (#2741), anti-phospho-AKT1 (Ser473) (#9018), anti-phospho-AKT2 (Ser474) (#8599), anti-FOXO1 (#9454), anti-FOXO3a (#12829) and anti-FOXO4 (#9472). Other antibodies used include: anti-phospho-AKT3 (Ser472) (#AP3468a, Abgent), anti-integrin β1 (#610467, BD transduction Laboratories), anti-β-actin (#A5441, Sigma-Aldrich), and anti-LAMC2 (#SC-28330, Santa Cruz). The AKT inhibitor MK-2206 was purchased from Selleckchem.

Rao G., Pierobon M., Kim I.K., Hsu W.H., Deng J., Moon Y.W., Petricoin E.F., Zhang Y.W., Wang Y, & Giaccone G. (2017). Inhibition of AKT1 signaling promotes invasion and metastasis of non-small cell lung cancer cells with K-RAS or EGFR mutations. Scientific Reports, 7, 7066.

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Protein Expression Analysis in Myocardial Samples

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Myocardial samples of 50 μg were separated in 10%–15% SDS-PAGE gels and were transferred onto nitrocellulose membranes. Following block, the membranes were incubated with the anti-Akt1, anti-Akt2, anti-Akt3, anti-AMPKα2, anti-p16, anti-p21, anti-Atg5, anti-Atg7, anti-Beclin1, anti-LC3B, anti-p62, anti-Parkin, anti-Bnip3, anti-PGC-1α, anti-TFEB (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-FundC1, anti-UCP2, anti-Pink1, (1:1000; Abcam, Cambridge, MA, USA), anti-GAPDH and anti-tubulin (loading controls) (1:1000; Cell Signaling Technology, Danvers, MA, USA) antibodies. After washing in Tris-buffered saline-Tween (TBST), nitrocellulose membranes were co-treated with horseradish peroxidase (HRP)-coupled secondary antibodies for 1 h at room temperature. A Bio-Rad Calibrated Densitometer was employed to scan the film and intensity of immunoblot bands was normalized to that of the loading control (GAPDH or α-tubulin) [16 (link)].

Wang S., Kandadi M.R, & Ren J. (2018). Double Knockout of Akt2 and AMPK Predisposes Cardiac Aging without Affecting Lifespan: Role of Autophagy and Mitophagy. Biochimica et biophysica acta. Molecular basis of disease, 1865(7), 1865-1875.

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Antibody-based immunoblotting protocol

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Sample preparation and immunoblotting were carried out as previously described^{25 (link)}. The following antibodies from Cell Signalling Technologies (MA, US) were used: anti-phospho-Stat3 (Tyr705: 9131), anti-total-Stat3 (9132), anti-phospho-Akt (Ser473: 9271), anti-total-Akt (9272), anti-phospho-Gsk3β (9331), anti-GSK3β (9315), anti-Akt1 (2967), anti-Akt2 (2964), anti-Akt3 (4059) and anti-Lamin A/C (2032). The following antibodies from Abcam (Cambridge, UK) were used: anti-cathepsin B (ab33538), anti-Lamp2 (ab13524), anti-Tubulin (ab6160) and anti-β-actin (ab8227). The following antibodies from Santa Cruz Biotechnology (CA, US) were used: anti-C/ebpα (sc-61), anti-histone H3 (sc8654), anti-Bax (sc-7480), anti-Bcl-2 (sc7382) and anti-Bcl-xL (sc-634). Other commercial antibodies used were: anti-cathepsin L (MAB9521), anti-cathepsin L (AF1515, used for the immunodetection of cathepsin L in the cathepsin L knockout and control glands) and anti-Bid (MB860) from R&D Systems (MN, US), anti-pan-p85 (Millipore, 06-496, also detects p50α/p55α subunits), anti-cytochrome c (65981A) and anti-E-cadherin (610182) from BD biosciences. All antibodies were used at a standard dilution of 1:1,000. Secondary horseradish peroxidase (HRP)-conjugated antibodies were purchased from Dako (Glostrup, Denmark).

Pensa S., Neoh K., Resemann H.K., Kreuzaler P.A., Abell K., Clarke N.J., Reinheckel T., Kahn C.R, & Watson C.J. (2014). The PI3K regulatory subunits p55α and p50α regulate cell death in vivo. Cell Death and Differentiation, 21(9), 1442-1450.

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