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P akt serine 473

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P-AKT Serine 473 is a lab equipment product that detects the phosphorylation of AKT at serine residue 473. Phosphorylation of AKT at this site is a key indicator of AKT activation, which plays a central role in various cellular processes such as cell growth, survival, and metabolism.

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Lab products found in correlation

β actin, by Merck Group (2 mentions) Rc dc assay, by Bio-Rad (2 mentions) Phospho perk, by Thermo Fisher Scientific (2 mentions) Gapdh, by Thermo Fisher Scientific (2 mentions) Actin antibody, by Merck Group (2 mentions) Serine p 2481 mtor, by Cell Signaling Technology (2 mentions) Serine 235 236 phospho s6 ribosomal protein, by Cell Signaling Technology (2 mentions) Sheep anti rabbit secondary antibodies, by GE Healthcare (2 mentions) Total mtor, by Cell Signaling Technology (2 mentions) Calnexin, by Cell Signaling Technology (1 mentions) Pgsk3β serine 9, by Cell Signaling Technology (1 mentions) Total oxphos human wb antibody cocktail, by Abcam (1 mentions) Bcl 2, by R&D Systems (1 mentions) P smad1 5 9, by Cell Signaling Technology (1 mentions) Smad1, by Cell Signaling Technology (1 mentions) Pan akt inhibitor gsk690693, by Selleck Chemicals (1 mentions) Anti ha, by Roche (1 mentions) Pd98059, by Merck Group (1 mentions) Ldn193189, by Merck Group (1 mentions) Ly294002, by Merck Group (1 mentions)

10 protocols using p akt serine 473

1

Immunoblot Analysis of Cell Signaling Pathways

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Immunoblot analysis was carried out as described.31 (link) Primary antibodies used were: Calnexin (#2433), p-cJUN serine 73 (#3270), cJUN (#9165), ERK (#9102), pAKT serine 473 (#4060), p-S6 (#4858) (all from Cell Signaling. 1:1000), and MAP3K1 (ab220416, Abcam; 1:100). Band intensities were quantitated using ImageJ. All pictured blots derive from the same experiment and were processed in parallel.
Nixon M.J., Formisano L., Mayer I.A., Estrada M.V., González-Ericsson P.I., Isakoff S.J., Forero-Torres A., Won H., Sanders M.E., Solit D.B., Berger M.F., Cantley L.C., Winer E.P., Arteaga C.L, & Balko J.M. (2019). PIK3CA and MAP3K1 alterations imply luminal A status and are associated with clinical benefit from pan-PI3K inhibitor buparlisib and letrozole in ER+ metastatic breast cancer. NPJ Breast Cancer, 5, 31.
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2

Molecular Signaling After SAH

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We performed immunohistochemistry staining according to the manufacturer's protocol of the kit. Rats were anesthetized and sacrificed by perfusion with 4% paraformaldehyde 24 hours after SAH induction or sham injury. The sections were incubated with the antibodies against pAkt (serine-473) (1∶1000, Cell signaling Technology, USA) and pGSK3β (serine-9) (1∶1000, Cell signaling Technology, USA).
Hong Y., Shao A., Wang J., Chen S., Wu H., McBride D.W., Wu Q., Sun X, & Zhang J. (2014). Neuroprotective Effect of Hydrogen-Rich Saline against Neurologic Damage and Apoptosis in Early Brain Injury following Subarachnoid Hemorrhage: Possible Role of the Akt/GSK3β Signaling Pathway. PLoS ONE, 9(4), e96212.
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3

Protein Expression Analysis by Capillary Electrophoresis

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Specific protein expression in cell lines was determined by Western blot analysis or capillary electrophoresis as described before. Capillary electrophoresis was run on the Wes instrument (Proteinsimple, CA). The following antibodies were used on the Wes instrument: p-Akt (serine 473) (1:25, CST, Cell Signaling Technology, Danvers, MA), Akt (1:50, CST), Mcl-1 (1:50; CST:), Bcl-2 (1:25; R&D Systems), BIM (1:25; CST), Bcl-xL (1:25; CST), c-myc (1:25, CST), Usp9X (1:25; CST), Noxa (1:25, clone 114C307; Calbiochem), p-Akt (1:25, CST), Akt (1:25, CST), p-AMPK (1:25, CST), AMPK (1:25, CST), PHGDH antibody (Novus, #NBP1–87311), PSAT1 Polyclonal Antibody (Invitrogen #PA5–22124), β-actin (1:250, clone AC15; Sigma Aldrich) and secondary HRP-linked antibodies were purchased from Santa Cruz Biotechnology Inc. For the expression levels of respiratory complexes, the Total OXPHOS Human WB Antibody Cocktail was used (Abcam, Cambridge, MA). Western blots were acquired, using the Azure (C300) imaging system (CCD – camera based).
Ishida C.T., Zhang Y., Bianchetti E., Shu C., Trang N.T., Kleiner G., Sanchez-Quintero M.J., Quinzii C.M., Westhoff M.A., Karpel-Massler G., Prabhu V.V., Allen J.E, & Siegelin M.D. (2018). Metabolic Reprogramming by Dual AKT/ERK Inhibition Through Imipridones Elicits Unique Vulnerabilities in Glioblastoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(21), 5392-5406.
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4

Evaluation of ER Stress Response

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Infected mammalian cells were lysed in radioimmunoprecipitation assay buffer with the addition of protease (Roche cOmplete), and phosphatase inhibitors (GB Sciences). Protein levels of lysates were determined using the BioRad DC/RC assay. Equal amounts of protein lysate were boiled with SDS load buffer. Equal amounts of protein were loaded and immunoblotting with, Thermo GAPDH antibody was used as a loading control. Antibodies used in assay are as follows: CHOP (Thermo MA1-250; 1:2,000), BiP (Protein Tech Group 11587-1-AP; 1:5,000), Phospho-Perk (Thermo MA5-15033; 1:1,000), GAPDH (Thermo MA5-15738; 1:10,000), PDI (Enzo ADI-SPA-891; 1:5,000), P115 (Lab generated; 1:2,500), p-AKT Serine 473 (Cell Signaling 9271S; 1:1,000).
Treacy-Abarca S, & Mukherjee S. (2015). Legionella suppresses the host unfolded protein response via multiple mechanisms. Nature Communications, 6, 7887.
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5

Endoglin and ALK1 Signaling Assay

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Matrigel matrix was obtained from Corning. Insulin, human recombinant, zinc solution was purchased from Thermo Fisher Scientific. TGF-β1 and BMP-9 were obtained from R&D Systems. Inhibitors of ALK1 (LDN193189), PI3K inhibitor (LY294002), Src family inhibitor (PP2), and MEK inhibitor (PD98059) were obtained from Sigma–Aldrich. Pan-Akt inhibitor (GSK690693) was purchased from Selleckchem. The following antibodies were all purchased from Cell Signaling: P-Smad1/5/9, Smad1, P-Akt serine 473, P-p42/44 MAPK, Src, and IR. β-actin was purchased from Sigma–Aldrich, anti-HA was from Roche, and Endoglin (P3D1), Endoglin (A-8), ALK1, and EPDR1 antibodies were purchased from Santa Cruz Biotechnology.
Ahmed T., Flores P.C., Pan C.C., Ortiz H.R., Lee Y.S., Langlais P.R., Mythreye K, & Lee N.Y. (2022). EPDR1 is a noncanonical effector of insulin-mediated angiogenesis regulated by an endothelial-specific TGF-β receptor complex. The Journal of Biological Chemistry, 298(9), 102297.
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6

Protein Expression and Signaling Analysis

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Harvested cells were lysed with RIPA buffer and quantified using a Qubit Protein Assay Kit (Invitrogen). Total cell lysates were subjected to SDS-PAGE and immunoblotting to detect exogenous Galanin with mouse monoclonal anti-GFP antibodies (Sigma-Aldrich, St. Louis, MO, USA). We also examined the levels of cleaved PARP and phosphorylated Akt (p-Akt serine 473; Cell Signaling Technology, Danvers, MA, USA). Polyclonal antibodies against phosphorylated Erk1/2(p-Erk1/2 threonine 202/ tyrosine 204), Erk1/2, cyclin D1 and horseradish peroxidase-linked secondary antibodies were also purchased from Cell Signaling Technology. Beta-actin was used as an internal protein loading control (Abcam, Cambridge, MA, USA). The results of western blotting analysis were demonstrated with representative examples from three independent experiments.
Yoon D., Bae K., Lee M.K., Kim J.H, & Yoon K.A. (2018). Galanin is an epigenetically silenced tumor suppressor gene in gastric cancer cells. PLoS ONE, 13(2), e0193275.
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7

Antibody Panel for Signaling Pathway Analysis

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A list of antibodies used in this study is provided in Table 1.

List of antibodies used in this study.

AntigeneManufacturerCat. No.Dilution
AktCell Signaling, Danvers, USA9272S1:1000
GAPDHCell Signaling, Danvers, USA2118S1:1000
Goat anti-Rabbit IgG AF-555ThermoFischer Scientific, DarmstadtA-214281:500
IgG isotype controlCell Signaling, Danvers, USA3900S0.1 µg (IP)
Jagged-1Cell Signaling, Danvers, USA70109S1:1000
Anti-Mouse-HRPCell Signaling, Danvers, USA7076S1:10000
pAkt Serine 473Cell Signaling, Danvers, USA9271S1:1000
pRhoA Serine 188Santa Cruz, Santa Cruz, USASc-329541:1000
Anti-Rabbit-HRPCell Signaling, Danvers, USA7074S1:5000
RhoASanta Cruz, Santa Cruz, USASc-4181:1000
sGCβ1Sigma-Aldrich, Munich, GermanyG4405

1:1000 (WB)

1:100 (Immunostaining)

TubulinDianova, Hamburg, GermanyDLN-099931:1000
VimentinCell Signaling, Danvers, USA5741S

1:1000 (WB)

1:100 (Immunostaining)

0.1 µg (IP)

P-Rex1Cell Signaling, Danvers, USA13168S1:1000 (WB)
Hildebrand S., Ibrahim M., Schlitzer A., Maegdefessel L., Röll W, & Pfeifer A. (2022). PDGF regulates guanylate cyclase expression and cGMP signaling in vascular smooth muscle. Communications Biology, 5, 197.
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8

Immunoblotting Analysis of UPR Markers

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Infected mammalian cells were lysed in RadioImmunoprecipitation Assay (RIPA) buffer with the addition of protease (Roche cOmplete), and phosphatase inhibitors (GB Sciences). Protein levels of lysates were determined using the BioRad DC/RC assay. Equal amounts of protein lysate were boiled with SDS load buffer. Equal amounts of protein were loaded and immunoblotting with, Thermo GAPDH antibody was used as a loading control. Antibodies used in assay are as follows: CHOP (Thermo MA1-250) (1:2000), BiP (Protein Tech Group 11587-1-AP)(1:5000), Phospho-Perk (Thermo MA5-15033)(1:1000), GAPDH (Thermo MA5-15738) (1:10000), PDI (Enzo ADI-SPA-891) (1:5,000), P115 (Lab generated) (1:2,500), p-AKT Serine 473 (Cell Signaling 9271S) (1:1000).
Treacy-Abarca S, & Mukherjee S. (2015). Legionella suppresses the host unfolded protein response via multiple mechanisms. Nature Communications, 6, 7887.
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9

Antibody Validation for Signaling Pathways

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The following antibodies were purchased from Cell Signaling: Total mTOR (Cat # 2983); Serine P-2481 mTOR (cat # 2976); S6 (Cat # 2217); Serine 235/236 Phospho-S6 ribosomal protein (Cat # 2211); Akt (Cat # 4691); Serine P-473 Akt (Cat # 4060). Actin antibody was obtained from Sigma (Cat # A2103). Sheep anti-rabbit secondary antibodies were obtained from (GE Health Care Bioscience, Corp, Piscataway, NJ).
Soliman G.A., Steenson S.M, & Etekpo A.H. (2016). Effects of Metformin and a Mammalian Target of Rapamycin (mTOR) ATP-Competitive Inhibitor on Targeted Metabolomics in Pancreatic Cancer Cell Line. Metabolomics : open access, 6(3), 183.
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10

Antibody Validation for mTOR and AKT Pathway

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Antibodies against the following proteins were purchased from Cell Signaling: total mTOR (catalog no. 2983), serine P-2481 mTOR (catalog no. 2976), S6 (catalog no. 2217), serine 235/236 phospho-S6 ribosomal protein (catalog no. 2211), total Akt (catalog no. 4691), serine P-473 Akt (catalog no. 4060), as we previously reported (25 ). The actin antibody was obtained from Sigma (catalog no. A2103). Sheep anti-rabbit secondary antibodies were obtained from GE Health Care Bioscience.
Soliman G.A., Shukla S.K., Etekpo A., Gunda V., Steenson S.M., Gautam N., Alnouti Y, & Singh P.K. (2020). The Synergistic Effect of an ATP-Competitive Inhibitor of mTOR and Metformin on Pancreatic Tumor Growth. Current Developments in Nutrition, 4(9), nzaa131.
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