Pakt1 ser 473

Manufactured by Cell Signaling Technology

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PAKT1 (Ser 473) is a laboratory reagent used to detect and quantify the phosphorylation of the serine 473 residue of the AKT1 protein. It is commonly used in research applications to study signal transduction pathways involving the AKT1 protein and its role in cellular processes such as cell growth, proliferation, and survival.

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16 protocols using pakt1 ser 473

RPPA-Based PI3K Pathway Characterization in TNBC PDX Models

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RPPA was performed as previously described 29 (link) for 48 of the 57 TNBC PDX (9 PDX were established after the RPPA analysis). We calculated a PI3K pathway score with normalized data to assess pathway activation. Scores were obtained by calculating the sum for positive protein components (PI3K p110 subunit β, p-AKT1 (Ser473), p-AKT1 (Thr308), p-4E-BP1, p-p70-S6 kinase, p-S6 ribosomal protein; Cell Signaling Technology®) and subtracting the negative components of the pathway (PTEN, Cell Signaling Technology®). Eleven PDX classified as unstable (UNS) in Lehmann's classification were excluded from the analysis shown in Figure 3B.

Coussy F., Lavigne M., de Koning L., Botty R.E., Nemati F., Naguez A., Bataillon G., Ouine B., Dahmani A., Montaudon E., Painsec P., Chateau-Joubert S., Laetitia F., Larcher T., Vacher S., Chemlali W., Briaux A., Melaabi S., Salomon A.V., Guinebretiere J.M., Bieche I, & Marangoni E. (2020). Response to mTOR and PI3K inhibitors in enzalutamide-resistant luminal androgen receptor triple-negative breast cancer patient-derived xenografts. Theranostics, 10(4), 1531-1543.

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Alteronol Induces Autophagy-Mediated Apoptosis

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Alteronol with 99% purity was obtained from Shantou Strand Bio Technology Co., Ltd. (Shantou, China). DMSO, 3-methyladenine (3-MA), and 2-mercaptoethanol, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma ((Shanghai, China). Annexin V-FITC and propidium iodide (PI) was obtained from BD Biosciences (San Jose, USA). Primary antibodies: Caspase3, Caspase9, AKT1, p-AKT1(Ser473), Beclin1, mTOR, p-mTOR(Ser2448), Bcl-2, p62, Bax, Smad3, phospho-Smad3 (Ser423/425), and LC3 were from Cell Signaling Technology (Danvers, MA, USA). The antibodies for β-actin, E-cadherin, and vimentin were from Santa Curz Biotechnology (Santa Cruz, CA).

Bao Y., Ding Z., Zhao P., Li J., Chen P., Zheng J, & Qian Z. (2020). Autophagy inhibition potentiates the anti-EMT effects of alteronol through TGF-β/Smad3 signaling in melanoma cells. Cell Death & Disease, 11(4), 223.

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Western Blot Analysis of Signaling Proteins

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Western blot analysis was performed using methods described previously^{30 (link)}. Antibodies against the following proteins were used: AKT1, pAKT1 (Thr308), pAKT1 (Ser 473), STAT3, p-Tyr705 STAT3, ERK, pERK, pALK and pGSK3α/β (Cell Signaling Technology, Beverly, MA, USA), JUNB and CJUN (Santa Cruz Biotechnology, Santa Cruz, CA, USA), AKT2 and AKT3 (Millipore, Billerica, Massachusetts, MA, USA). β-Actin (Sigma, St Louis, MO) served as a control for protein load and integrity in all immunoblots.

Atsaves V., Zhang R., Ruder D., Pan Y., Leventaki V., Rassidakis G.Z, & Claret F.X. (2015). Constitutive control of AKT-1 gene expression by JUNB / CJUN in ALK+ anaplastic large cell lymphoma: a novel crosstalk mechanism. Leukemia, 29(11), 2162-2172.

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Quantifying Myocardial Protein Levels

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Samples obtained from the remote zone at week 4 after infarction. The primary antibodies were resistin (Chemicon), p-Akt1 (ser473, Cell Signaling Technology), Akt1 (Santa Cruz Biotechnology), NGF (Chemicon) and β-actin (Santa Cruz Biotechnology). For a detailed method, please refer to the Supplementary material online.

Lee T.M., Chen W.T, & Chang N.C. (2016). Sitagliptin decreases ventricular arrhythmias by attenuated glucose-dependent insulinotropic polypeptide (GIP)-dependent resistin signalling in infarcted rats. Bioscience Reports, 36(2), e00307.

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Western Blot Analysis of Protein Lysates

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Protein lysates were collected and analyzed by western blot as previously described^{24 (link)}. In brief, primary antibodies against GAPDH (Cell Signaling, 5174), Lamin A/C (Cell Signaling, 4777), p-Akt1 Ser 473 (Cell Signaling, 4060), total Akt1 (Cell Signaling, 4691), IFIT2 (Abcam - ab113112), Viperin (Cell Signaling -13996 S), Actinin 2 (Thermo Fisher Scientific - PA5-27863), Alpha-Pix (Cell Signaling -4753 S), RVFV Nucleoprotein (BEI Resources, NR-43188), or HRP-conjugated actin (Abcam, ab49900) were diluted in 3% milk solution per the manufacturer’s recommended dilutions followed by the addition of the appropriate secondary antibody. Cropped, representative images are shown in main figures, but uncropped originals can be found in the Supplemental Figure 8.

Pinkham C., Dahal B., de la Fuente C.L., Bracci N., Beitzel B., Lindquist M., Garrison A., Schmaljohn C., Palacios G., Narayanan A., Campbell C.E, & Kehn-Hall K. (2017). Alterations in the host transcriptome in vitro following Rift Valley fever virus infection. Scientific Reports, 7, 14385.

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Protein Expression Analysis Protocol

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Antibodies against CHOP, IRE1α, Calnexin, LC3, GM130, p-Akt1(Thr308) and p-Akt1(Ser473) were purchased from Cell Signaling Technology (Boston, MA, USA). ERGIC3, p62 and MMP-9 antibodies were obtained from Abcam (Beverly, MA, USA). Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Akt1 were purchased from AbFrontier (Seoul, Korea). Antibodies against PCNA, VEGF, cyclin B1 and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Tunicamycin and chloroquine were purchased from Sigma (St. Louis, MO, USA). TUDCA was purchased from EMD Chemicals (Gibbstown, NJ, USA).

Hong S.H., Chang S.H., Cho K.C., Kim S., Park S., Lee A.Y., Jiang H.L., Kim H.J., Lee S., Yu K.N., Seo H.W., Chae C., Kim K.P., Park J, & Cho M.H. (2016). Endoplasmic reticulum-Golgi intermediate compartment protein 3 knockdown suppresses lung cancer through endoplasmic reticulum stress-induced autophagy. Oncotarget, 7(40), 65335-65347.

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Comprehensive AKT Pathway Analysis

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Antibodies against pan-AKT (#4691), phospho-AKT Thr308/9 (#2965), phospho-AKT Thr450/1 (#9267), phospho-AKT Ser473/4 (#9271), AKT1 (#2938), pAKT1 Ser473 (#9018), AKT2 (#3063), phospho-AKT2 (pAKT2) Ser474 (#8599), AKT3 (#14982), and ERK (#4695) were from Cell Signaling Technology (Beverly, MA).

Wang J., Zhao W., Guo H., Fang Y., Stockman S.E., Bai S., Ng P.K., Li Y., Yu Q., Lu Y., Jeong K.J., Chen X., Gao M., Liang J., Li W., Tian X., Jonasch E., Mills G.B, & Ding Z. (2018). AKT isoform-specific expression and activation across cancer lineages. BMC Cancer, 18, 742.

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Western Blot Analysis of Signaling Pathways

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Cells or bone tissues were lysed with lysis buffer (200 mM Tris-HCl (pH 7.5), 1.5 M NaCl, 10 mM EDTA, 25 mM sodium pyrophosphate, 10 mM glycerolphosphate, 10 mM sodium orthovanadate, 50 mM NaF, 1 mM PMSF, 0.5% Triton, in combination with protein inhibitor cocktail). Twenty micrograms of protein lysates of each sample was subjected to SDS-PAGE analysis and transferred onto nitrocellulose membranes. Blots were incubated with the specific primary antibodies overnight at 4°C. After being washed three times for 15 min each with TBST (TBS+0.1 % Tween 20), blots were incubated with horseradish peroxidase–conjugated secondary antibody (Sigma) and visualized by chemiluminescence (Protein Simple). The following antibodies were used: p-Smad1/5/8 (Cell Signaling; 9511s), Smad1 (Cell Signaling; 9743), NF-κB p65 (Cell Signaling; 4767), IκBα (Cell Signaling; 4814), p38α MAPK (Cell Signaling; 9212), p-p38MAPK (T180/182) (Cell Signaling; 9211), p-Akt1 (Ser473) (Cell Signaling; 9271s), Akt1 (Cell Signaling; 9272), β-Catenin (BD; 610153), Smad2/3(Cell Signaling; 3102), p-Smad2 (S465/467) (Cell Signaling; 3101s), p-Smad3 (S423/425) (Cell Signaling; 9520s), and β-actin (Santa Cruz; sc-81178).

Li A., Cong Q., Xia X., Leong W.F., Yeh J., Miao D., Mishina Y., Liu H, & Li B. (2017). Pharmacologic Calcitriol Inhibits Osteoclast Lineage Commitment via the BMP-Smad1 and IκB-NF-κB Pathways. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 32(7), 1406-1420.

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Dissecting AKT1 Signaling Pathway in Cell Cultures

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MK-2206, LY294002, BAY 11-7082, and CP-673451 were purchased from Selleck Chemicals (Houston, TX, USA). 666-15 was obtained from Tocris Bioscience (Bristol, UK). Puromycin was purchased from Solarbio (Beijing, China). Mouse PDGFA factor from Novus Biologicals (Colorado, USA). pLXIN-mutAKT1 (AKT1E17K), pLXIN-myrAKT1 (myristoylated AKT1) expression plasmids, and the empty control pLXIN vector have been described previously^{14 (link)}. The CREB S133A cDNA was amplified using pCF-CREB M1 plasmid (#22969, Addgene, Watertown, MA, USA) as templates and subcloned into a pLXIN retroviral vector. pGL3-Basic and pRL-TK plasmid were from Promega (Madison, WI, USA). PTEN (#9559), PDGFRα (#3174), PDGFRβ (#4564), p-AKT (Ser-473) (#4060), AKT1 (#2967), p-AKT1 (Ser-473) (#9018), CREB (#9197), p-CREB (Ser133) (#9198), p-PDGFRα^Y849/PDGFRβ^Y857 (#3170), cleaved caspase-3 (#9664), AKT2 (#3063), AKT3 (#3788), p-IκBα (#2859), IκBα (#4814), FOXO1 (#2880), FOXO3a (#2497), LaminB1 (#13435), Ki-67 (#12202), GAPDH (#2118) and β-actin (#4970) antibodies were from Cell Signaling Technology (Danvers, MA, USA). All horseradish peroxidase (HRP)-labeled secondary antibodies were from Jackson Immuno Research (West Grove, PA, USA).

Wan X., Zhou M., Huang F., Zhao N., Chen X., Wu Y., Zhu W., Ni Z., Jin F., Wang Y., Hu Z., Chen X., Ren M., Zhang H, & Zha X. (2021). AKT1-CREB stimulation of PDGFRα expression is pivotal for PTEN deficient tumor development. Cell Death & Disease, 12(2), 172.

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Western Blot Analysis of Signaling Proteins

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