Phospho akt ser473

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Phospho-Akt (Ser473) is a primary antibody that specifically recognizes Akt (also known as Protein Kinase B) phosphorylated at serine 473. Akt is a key regulator of various cellular processes, including cell survival, proliferation, and metabolism.

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647 protocols using phospho akt ser473

Western Blot Analysis of Cell Signaling Proteins

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Proteins from total cell lysates were separated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and probed with different primary antibodies against phospho (p)‐AKT (Ser473; Cell Signaling Technology), AKT (Cell Signaling), p‐GSK‐3β (Ser256; Cell Signaling Technology), GSK‐3β (Cell Signaling Technology), Nrf2 (Abcam), HO‐1 (heme oxygenase‐1; Cell Signaling Technology), NQO‐1 (NAD(P)H quinone dehydrogenase 1; Cell Signaling) and GAPDH (glyceraldehyde‐3‐phosphate dehydrogenase; Abcam). Of note, we only showed cleaved‐caspase 3 bands in figures instead of total caspase 3 and cleaved‐caspase 3 bands in the same membrane; the reason for this is that when we exposure total‐caspase 3 and cleaved‐caspase 3 at the same time, the cleaved‐caspase 3 band was undetectable, which has been described in our previous study.¹²

Yu H., Zhen J., Yang Y., Du J., Leng J, & Tong Q. (2020). Rg1 protects H9C2 cells from high glucose‐/palmitate‐induced injury via activation of AKT/GSK‐3β/Nrf2 pathway. Journal of Cellular and Molecular Medicine, 24(14), 8194-8205.

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Platelet activation signaling pathways

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MRS2395 [2,2-Dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propyl
ester], adenosine diphosphate (ADP), wortmannin, thrombin and all other chemicals and reagents were purchased from Sigma-Aldrich
(St Louis, MO, USA) or previously mentioned sources unless specified otherwise.[12 (link), 13 (link)] TRAP-6 (SFLLRN), U73122, U73343, Ro 31–8220, Rottlerin, Go6976, MRS2179, BAPTA,
PSB 0739 and AR-C 66096 were obtained from Tocris (Bristol, UK). Ticagrelor was purchased from Oxchem Corporation (Wood Dale, IL,
USA). AYPGKF-NH₂ was obtained from Abgent (San Diego, CA, USA). Oregon Green® 488 BAPTA-1 AM (OG488 BAPTA-1 AM)
was from Thermo Fisher Scientific (Carlsbad, CA, USA). PAC-1-FITC and anti-CD62P-APC were obtained from BD Bioscience (San Jose,
CA, USA). Anti-AKT, phospho (p)AKT-Ser473, pGSK3β-Ser9 and pPKC-Ser substrates were from Cell Signaling Technology
(Danvers, MA, USA).

Mitrugno A., Rigg R.A., Laschober N.B., Ngo A.T., Pang J., Williams C.D., Aslan J.E, & McCarty O.J. (2017). Potentiation of TRAP-6-induced platelet dense granule release by blockade of P2Y12 signaling with MRS2395. Platelets, 29(4), 383-394.

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Western Blot Analysis of Cellular Proteins

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Proteins were separated on a 10% SDS-PAGE gels, transferred to PVDF membrane (polyvinylidenedifluoride, Millipore, USA), and incubated overnight at 4 °C with antibodies directed against SIRT3, SOD2, Akt, phospho(p)-Akt (Ser 473) (1:1,000, Cell Signaling Technology, USA), acetylated(Ac)-SOD2 (Lys68) antibody (1:1,000, Abcam, USA), p-eNOS (Ser 1177), eNOS (1:1,000, BD Biosciences, USA) or β-actin (1:1,000, Santa Cruz, USA). After washing blots to remove excessive primary antibody binding, blots were incubated for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000, Boster, China) at room temperature. The blots were developed with an enhanced chemiluminescence detection kit (Roche, USA). The immunoblot was visualized with ChemiDocXRS (Bio-Rad, Hercules, USA) and the blot densities were analyzed with Lab Image software.

Yang L., Zhang J., Xing W., Zhang X., Xu J., Zhang H., Chen L., Ning X., Ji G., Li J., Zhao Q, & Gao F. (2016). SIRT3 Deficiency Induces Endothelial Insulin Resistance and Blunts Endothelial-Dependent Vasorelaxation in Mice and Human with Obesity. Scientific Reports, 6, 23366.

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Protein Extraction and Western Blot Analysis

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One hundred mg of adipose, liver, or intestine tissue was homogenized in a commercial PRO-PREP Protein Extraction Solution (Beyotime, China). Total protein lysates were fractionated by 10% SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes (Immobilon™-P; Merck Millipore, USA). Afterwards, the membranes were blocked with 5% nonfat milk for 1 h at room temperature in TBST buffer (10 mM Tris, 150 mM NaCl, pH 7.6, and 0.1% Tween 20) and probed with primary antibodies overnight at 4°C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody. The dilutions of primary and secondary antibodies have been described in Section 2.12. Protein bands were developed using an enhanced chemiluminescence reagent (Merck Millipore). The blots were probed with the primary antibodies against GAPDH (Bioworld Technology, Inc., China), TLR4, IKB-α, NF-κB (Abcam, USA), AKT, phospho- (p-) AKT (Ser473), GSK3β, and p-GSK3β (Ser9) (Cell Signaling Technology, USA).

Wei Z., Shen P., Cheng P., Lu Y., Wang A, & Sun Z. (2020). Gut Bacteria Selectively Altered by Sennoside A Alleviate Type 2 Diabetes and Obesity Traits. Oxidative Medicine and Cellular Longevity, 2020, 2375676.

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Western Blot and Immunoprecipitation Protocols

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Western blot and immunoprecipitation protocols have been described before [17 (link)]. Antibodies anti-cleaved caspase-3, γ-H2AX, platelet-derived growth factor receptor (PDGFR) α, PDGFRβ, vascular endothelial growth factor receptor (VEGFR) 2, phospho (P)-AKT ser 473, P-p65 ser 536, P-IκBα ser 32, P-p42/44 MAPK Thr202/Tyr204 and PTEN were purchased from Cell Signaling (Beverly, MA, USA). The antibodies anti-KIT and anti-tubulin were from Dako (Glostrub, Denmark) and Sigma-Aldrich (St Louis, MO, USA) respectively.

Wang E, & Sorolla A. (2019). Sensitizing endometrial cancer to ionizing radiation by multi-tyrosine kinase inhibition. Journal of Gynecologic Oncology, 31(3), e29.

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Western Blot Analysis of RBM20 and Signaling

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Total protein was separated by SDS-PAGE gel, and transferred onto a PVDF membrane. The membrane was probed with antibodies against RBM20 (home-made) which was validated in our previous studies [9 , 18 (link), 27 (link), 43 (link)], Akt (Cell Signaling), phospho(p)-Akt(Ser473) (Cell Signaling), p70S6K1(Cell Signaling), phosphor(p)-p70S6K1 (Thr389) (Cell Signaling), 4E-BP1 (Cell Signaling). Rabbit anti-mouse IgG-conjugated with horseradish peroxidase (Fisher Scientific) was served as the secondary antibody. The blot was developed with ECL western blotting substrate (Bio-Rad) and exposed to CL-Xposure film (Thermo Scientific). Anti-Histone3 (Cell Signaling) and GAPDH (Santa Cruz) were served as the protein loading control.

Zhu C., Yin Z., Tan B, & Guo W. (2017). Insulin regulates titin pre-mRNA splicing through the PI3K-Akt-mTOR kinase axis in a RBM20-dependent manner. Biochimica et biophysica acta, 1863(9), 2363-2371.

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P2X7 Signaling Regulates Cancer Stem Cells

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P2X7 agonist benzoyl ATP (BzATP), P2X7 antagonist A740003, and PI3K antagonist LY294002 were purchased from Sigma‐Aldrich (St. Louis, MO). Akt, phospho (p)‐Akt (Ser473), GSK3β, p‐GSK3β (Ser9), mTOR, p‐mTOR (Ser2448), HIF1α, β‐catenin, TCF1 and lamin B1 rabbit monoclonal antibodies (Abs) were from Cell Signaling Technologies Inc. (Milan, Italy). P2X7, E‐cadherin, fibronectin, vimentin, Snail, ALDH1A1, CD31, Ki67 and PCNA rabbit monoclonal Abs were purchased from Abcam (Cambridge, UK). CD133 antibody was from Miltenyi Biotec (Teterow, Germany).

Zhang Y., Cheng H., Li W., Wu H, & Yang Y. (2019). Highly‐expressed P2X7 receptor promotes growth and metastasis of human HOS/MNNG osteosarcoma cells via PI3K/Akt/GSK3β/β‐catenin and mTOR/HIF1α/VEGF signaling. International Journal of Cancer, 145(4), 1068-1082.

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Comprehensive Western Blot Analyses

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For western blot, samples were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with various antibodies; anti-GAPDH (#2118), N-cadherin (#13116), E-cadherin (#3195), Vimentin (#5741), Slug (#9585), Snail-1 (#3879), ZEB1 (#3396), ZO-1 (#8193), phospho-Met (Tyr1349) (#3121), Ron (#4269), p β-catenin (Thr41 or Ser45) (#9565), β-catenin (#8480), S6 (#2217), total Akt (#9272), phospho-c-Myc (Thr58/Ser62) (#9401), Ki-67 (#9027), phospho-AKT (Ser473) (#9271), phospho-EGFR (Tyr1068) (#11862), LRIG1 (#12752), c-Met (#8198) and antibodies were purchased from Cell Signaling Technology. LRIG1 (G-20) (#sc-50075) and EGFR (#sc-03) antibodies were purchased from Santa Cruz Biotechnology. c-Myc antibody (clone 9E11) (#MS-127-P0) was purchased from Neomarkers. Fibronectin (#GTX112794), CD44 (#GTX102111) and Twist (#GTX127310) were purchased from Genetex (CA, USA). Actin (#A5441) and Tubulin (#T5168) were purchased from Sigma (MO, USA). All antibodies used horseradish peroxidase-conjugated secondary antibodies (Biorad), followed by developing with SuperSignal West chemicals (Pierce). An AlphaInnotech imaging station with FluorChem software was used to capture images. All data are representative of more than three independent experiments.

Yokdang N., Hatakeyama J., Wald J.H., Simion C., Tellez J.D., Chang D.Z., Swamynathan M.M., Chen M., Murphy W.J., Carraway KL I.I.I, & Sweeney C. (2015). LRIG1 opposes epithelial to mesenchymal transition and inhibits invasion of basal-like breast cancer cells. Oncogene, 35(22), 2932-2947.

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Anticancer Drug Screening in Cell Lines

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MiaPaCa2, MDA-MB231, Panc1, AsPC1, and HEK293T cell lines were obtained from American Type Culture Collection (ATCC) and mycoplasma-free. These cells were cultured in Dulbecco’s minimal essential medium (DMEM) from Nacalai (California, USA) supplemented with 10% v/v FBS and penicillin (100 U/mL)/streptomycin (100 μg/mL) from Hyclone (IL, USA). Antibodies for GAPDH (14C10, #2118), phospho-AKT Ser473 (#9271), phospho-ERK Thr202/Tyr204 (#9101), phospho-S6 Ser235/236 (#2211), YAP (#4912), phospho-YAP Ser127 (#4911), and phospho-LATS1 Ser909 (#9157) were from Cell Signaling Technology (MA, USA). Antibody for TAZ (#HPA007415) was from Sigma-Aldrich (MO, USA). Gemcitabine-HCl (#S1149) was obtained from Selleck Chemicals (TX, USA), while doxorubicin-HCl (#D-4000) from LC Laboratories (MA, USA). PD184352, Triciribine and Rapamycin were obtained from Sigma-Aldrich (MO, USA). The Cell viability was assayed by colorimetric based CellTiter 96^® AQueous One Solution Cell Proliferation kit (#G3581, Promega), per manufacture’s protocol.

Chai T.F., Manu K.A., Casey P.J, & Wang M. (2020). Isoprenylcysteine carboxylmethyltransferase is required for the impact of mutant KRAS on TAZ protein level and cancer cell self-renewal. Oncogene, 39(31), 5373-5389.

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Western Blot Protocol for Protein Analysis

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Cell lysates were prepared by washing once with cold PBS followed by lysis with a modified RIPA buffer containing 25 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 1 % NP-40, 10 % glycerol, 2 mM Na₃VO₄, and 1X EDTA-free protease inhibitor cock-tail tablets (Roche) as previously described by Chinn et al. (2011) (link). Protein concentration was determined using the Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s protocol. SDS-PAGE was performed with 10–25 μg of protein loaded for each sample. Protein was transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) and probed overnight at 4 °C with the following primary antibodies: phospho-AKT (Ser473) (#4060), AKT (#9272), phospho-ERK1/2 (Thr202/Tyr204) (#4370), ERK1/2 (#4696), EGFR (C74B9, #2646), BIM (#2819), cleaved caspase-3 (#9661; Cell Signaling Technology, Danvers, MA, USA), phospho-EGFR (Tyr1068; #44788G; Invitrogen, Carlsbad, CA, USA), PARP-1 (#sc-8007; Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-actin (#A2228; Sigma-Aldrich, St. Louis, MO, USA). Blots were then incubated for 1 h at room temperature with the HRP-conjugated secondary antibodies, anti-mouse IgG (W4021) and anti-rabbit IgG (W4011; Promega, Madison, WI, USA), and visualized by chemiluminescence with Amersham ECL (GE Healthcare, Buckinghamshire, UK).

Holland W.S., Chinn D.C., Lara PN J.r., Gandara D.R, & Mack P.C. (2014). Effects of AKT inhibition on HGF-mediated erlotinib resistance in non-small cell lung cancer cell lines. Journal of cancer research and clinical oncology, 141(4), 615-626.

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