Rabbit anti phospho akts473

Manufactured by Cell Signaling Technology

Sourced in United States

Rabbit anti-phospho-AktS473 is a primary antibody that specifically recognizes the phosphorylated form of the serine 473 residue on the Akt protein. This antibody can be used to detect and quantify the activation of the Akt signaling pathway in various biological samples.

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Lab products found in correlation

Rabbit anti phospho akt t308, by Cell Signaling Technology (4 mentions) Rabbit anti akt, by Cell Signaling Technology (4 mentions) Rabbit anti erk1 2, by Cell Signaling Technology (3 mentions) Mouse anti erk1 2, by Cell Signaling Technology (2 mentions) Rabbit anti phospho erk1 2 t202 y204, by Cell Signaling Technology (2 mentions) Ecl detection system, by Thermo Fisher Scientific (1 mentions) Rabbit anti gapdh, by Cell Signaling Technology (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Bradford reagent, by Bio-Rad (1 mentions) Coomassie plus protein assay reagent, by Thermo Fisher Scientific (1 mentions) C digit blot scanner, by LI COR (1 mentions) Supersignal west femto maximum sensitivity substrate kit, by Thermo Fisher Scientific (1 mentions) Immobilon polyvinylidene difluoride pvdf membrane, by Merck Group (1 mentions) Sds page, by Bio-Rad (1 mentions) Mouse anti gapdh antibody, by Merck Group (1 mentions) Sheep anti mouse igg, by GE Healthcare (1 mentions) Blotting grade blocker, by Bio-Rad (1 mentions) Donkey anti rabbit igg, by GE Healthcare (1 mentions) Amersham ecl, by GE Healthcare (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions)

12 protocols using rabbit anti phospho akts473

Western Blot Analysis of AKT Phosphorylation

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Cells were lysed in ice-cold lysis buffer (25 mM Tris pH 8.0; 1.5 mM EGTA, 0.5 mM EDTA, protease inhibitor cocktail, and Triton X-100 1%). The total protein concentration was determined with the Bradford reagent (Bio-Rad: Hercules, CA, USA). Equivalent amounts of proteins (15 μg/well) were then separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). The membranes were subsequently immunoblotted with the appropriate primary antibody at 4 °C overnight (rabbit anti-AKT (1:4000, Cell Signaling # 4691); rabbit anti-phospho AKT S473 (1:4000, Cell signaling # 9271); rabbit anti-phospho AKT T308 (1:4000, Cell signaling # 9275)) and then incubated with HRP conjugated secondary anti-mouse or anti-rabbit antibody (1:4000, Jackson Immuno Research: West Grove, PA, USA). Antibody-antigen complexes were visualized by detecting enhanced chemiluminescence using the ECL detection system (Thermo Scientific: Waltham, MA, USA) on digital images (Bio-Rad). Equal protein loading was assessed by the expression of GAPDH (rabbit anti-GAPDH (1:4000, Cell signaling # 5174)). Total and phosphorylated protein expression were determined in different blots in the same running.

Rorato R., Borges B.D., Uchoa E.T., Antunes-Rodrigues J., Elias C.F, & Elias L.L. (2017). LPS-Induced Low-Grade Inflammation Increases Hypothalamic JNK Expression and Causes Central Insulin Resistance Irrespective of Body Weight Changes. International Journal of Molecular Sciences, 18(7), 1431.

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Western Blot Analysis of PTEN and Akt

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Cells were lysed in RIPA buffer (50mM Tris-HCl, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, PH 8.0) supplemented with a protease inhibitor cocktail (Roche Applied Science). The protein concentration was measured using a Bradford protein assay kit (Coomassie Plus Protein Assay reagent, Thermo). Protein samples were separated by 10% SDS-PAGE (Bio-Rad) and electroblotted onto 0.45μm Immobilon polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked with 5% Blotting-Grade Blocker (Bio-Rad) in PBST for 1 h at room temperature. The membrane was incubated with the respective antibodies: rabbit anti-PTEN XP (1:1000; Cell signaling,), rabbit anti-phospho-Akt (S473, 1:2,000; cell signaling), rabbit anti-Akt1 (1:500; Thermo) and mouse anti-GAPDH antibody (1:4,000; Millipore) overnight at 4 °C. Membranes were then incubated for 1 h at room temperature with Amersham ECL peroxidase-lined secondary antibodies: sheep anti-mouse IgG (1:10,000, GE Healthcare) or donkey anti-rabbit IgG (1:10,000, GE Healthcare). Western blot immunoreactivity was detected using a Super Signal West Femto Maximum Sensitivity Substrate Kit (Thermo) in C-DiGit Blot Scanner (LI-COR Biosciences). The density of protein bands was measured using Image J software, and values were normalized to the densitometric values of GAPDH or total Akt1 in samples.

Zhang L., Zhou M., Qin G., Weintraub N.L, & Tang Y. (2014). MiR-92a regulates viability and angiogenesis of endothelial cells under oxidative stress. Biochemical and biophysical research communications, 446(4), 952-958.

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Lung Cancer Tissue Microarray Protocol

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Recombinant human APRIL was purchased from ProSpec-TanyTechnoGene (East Brunswick, NJ, USA). Antibodies against APRIL (ab64967), BCMA (ab5972), and TACI (ab64967) were purchased from Abcam (Cambridge, MA, USA). Rabbit anti-ERK1/2, mouse anti-ERK1/2 and rabbit anti-phospho-Akt S473 antibodies were from Cell Signaling Technology (Danvers, MA, USA). Other reagents were from Sigma-Aldrich (St. Louis, MO, USA), except where specifically indicated. The lung cancer tissue microarray was obtained from Shanghai Zhuoli Biotechnology Co., Ltd. (Shanghai, China).

Dou H., Yan Z., Zhang M, & Xu X. (2017). APRIL promotes non-small cell lung cancer growth and metastasis by targeting ERK1/2 signaling. Oncotarget, 8(65), 109289-109300.

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Protein Extraction and Western Blot Analysis

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Whole‐cell extracts were prepared by lysing cells with RIPA buffer (50 mM Tris‐HCl [7.4], 150 mM NaCl, 1% Triton X‐100, 0.5% sodium deoxycholate, 0.1% SDS) containing 5 mM sodium vanadate, 5 mM NaF, and complete EDTA‐free protease inhibitor (Roche) for 30 minutes at 4°C. Insoluble materials were removed by centrifugation. Western blotting was carried out using standard methods. The following antibodies were used: rabbit anti‐phospho‐EGFR (Y1068) (1:3000; Cell Signaling #2237), mouse anti‐EGFR (1:3000; Cell Signaling #2239), rabbit anti‐phospho‐AKT (S473) (1:3000; Cell Signaling #9271), rabbit anti‐AKT (1:3000; Cell Signaling #9272), rabbit anti‐phospho‐ERK1/2 (T202/Y204) (1:3000; Cell Signaling #9101), rabbit anti‐ERK1/2 (1:10 000; Santa Cruz sc‐93), mouse anti‐actin (1:1000; Sigma C7207), anti‐mouse IgG‐HRP (1:10 000; GE Healthcare NA9310), and anti‐rabbit IgG‐HRP (1:10 000; GE Healthcare NA9340). Blots were developed using the ECL Western Blotting Detection Reagents (GE Healthcare RPN2109) and detected using the LAS‐3000 imaging system (Fujifilm).

Nishiya N., Oku Y., Ishikawa C., Fukuda T., Dan S., Mashima T., Ushijima M., Furukawa Y., Sasaki Y., Otsu K., Sakyo T., Abe M., Yonezawa H., Ishibashi F., Matsuura M., Tomida A., Seimiya H., Yamori T., Iwao M, & Uehara Y. (2021). Lamellarin 14, a derivative of marine alkaloids, inhibits the T790M/C797S mutant epidermal growth factor receptor. Cancer Science, 112(5), 1963-1974.

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Western Blot Analysis of Cell Signaling

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Protein lysates were obtained from cells or excised tumors from transplanted mice through solubilization in lysis buffer [PBS; 1% Sodium Dodecyl Sulfate (SDS; Sigma-Aldrich 05030); PhosSTOP™ (Roche 04 906 845 001); cOmplete™ Protease Inhibitor Cocktail (Roche 11873580001)], separated on 8–15% SDS-PAGE in reducing conditions and transferred onto nitrocellulose membrane (GE Healthcare 10600002). Western blot (WB) analyses were performed following standard protocols. The following Abs were used: rat ant-IRF4 (1:500; Biolegend, 646402); rabbit anti-BLIMP1 (1:1000; Cell Signaling, 9115); rabbit anti-phospho eIF2α (1:1000; Cell Signaling, 3398); rabbit anti-eIF2α tot (1:1000; Cell Signaling, 5324); rabbit anti-phospho S6 Ribosomal Protein (1000; Cell Signaling, 5364); rabbit anti-S6 Ribosomal Protein (1000; Cell Signaling, 2317); rabbit anti-LC3B polyclonal Ab (1:500; Novus Biologicals, NB100–220); rabbit anti-phospho AKT (S473) (1:1000; Cell Signaling, 9271); rabbit anti-phospho AKT (T308) (1:1000; Cell Signaling, 2965) rabbit anti-Akt (pan) (C67E7) (1:1000; Cell Signaling, 4691); rabbit anti-phospho BAD (1:1000; Cell Signaling, 4366); rabbit anti-BAD tot (1:1000; Cell Signaling, 9239); mouse anti-ACTB/b actin (1:5000; Sigma-Aldrich, A5441). Quantifications were obtained with the gel analysis option of ImageJ software.

Trudu M., Oliva L., Orfanelli U., Romano A., Di Raimondo F., Sanvito F., Ponzoni M, & Cenci S. (2022). Preclinical evidence of a direct pro-survival role of arginine deprivation in multiple myeloma. Frontiers in Oncology, 12, 968208.

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Comprehensive Antibody Panel for Cell Signaling

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The following primary antibodies were obtained from Cell Signaling Technology (catalog number, dilution in OBB): rabbit anti-phospho-AKT T308 (13038, 1:400), rabbit anti-phospho-AKT S473 (4060, 1:200), rabbit anti-phospho-Chk1 S317 (12302, 1:800), rabbit anti-phospho-Chk2 T68 (2661, 1:100), rabbit anti-phospho-4E-BP1 T37/46 (2855, 1:200), rabbit anti-phospho-S6 S235/236 (4858, 1:1000), rabbit anti-phospho-H2A.X S139 (“γH2A.X”) (9718, 1:400), rabbit anti-phospho-Histone H3 S10 (3377, 1:800), mouse anti-β-tubulin (86298, 1:400), rabbit anti-β-tubulin (2128, 1:400), mouse anti-BrdU (Bu20a) (5292, 1:200), rabbit anti-phospho-GSK3B S9 (5558, 1:400), rabbit anti-FOXO3 (2497, 1:200), rabbit anti-p21 (2947, 1:400), rabbit anti-p27 (3686, 1:400), and rabbit anti-phospho-Rb S807/811 (8516, 1:800). The following primary antibodies were obtained from Abcam (catalog number, dilution in OBB): rabbit anti-cyclin D1 (ab134175, 1:100), rabbit anti-geminin (ab195047, 1:100), and rabbit anti-cyclin A2 (ab181591, 1:500). Rabbit anti-phospho-RPA S4/8 antibodies were obtained from Bethyl (A300-245A, 1:400), rabbit anti-γ-tubulin antibodies from Sigma (T5192, 1:400), and Human Antibody against Centromere (CREST) antibodies from ImmunoVision (HCT-0100, 1:1000).

Chopra S.S., Jenney A., Palmer A., Niepel M., Chung M., Mills C., Sivakumaren S.C., Liu Q., Chen J.Y., Yapp C., Asara J.M., Gray N.S, & Sorger P.K. (2019). Torin2 exploits replication and checkpoint vulnerabilities to cause death of PI3K-activated triple-negative breast cancer cells. Cell systems, 10(1), 66-81.e11.

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Immunoblotting and Antibody Validation

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S1P was purchased from Cayman
Chemicals and JTE-013 from Sigma-Aldrich. Mouse anti-ERK 1/2 (1:1000,
clone 3A7), rabbit anti-phospho-ERK1/2 (T202/Y204) (1:1000, clone
D13.14.4E), rabbit anti-GFP (1:200, clone D5.1), rabbit anti-HA tag
(1:1000, clone C29F4), rabbit anti-p38 (1:1000, catalog no. 9212),
rabbit anti-phospho-p38 (T180/Y192) (1:1000, clone D3F9), rabbit anti-AKT
(1:1000, clone C67E7), and rabbit anti-phospho-AKT (S473) (1:1000,
clone D9E) antibodies were obtained from Cell Signaling. The mouse
anti-myc antibody (1:800, clone 9E10) was from Santa Cruz Biotechnology,
and the mouse anti-HA antibody (1:1000, clone 12CA5) was from Roche.
Rabbit anti-Flag (1:1000, clone F-7425), mouse anti-β-actin
(1:10000, clone A1978), and rabbit anti-β-catenin (1:1000, clone
C2206) antibodies were from Sigma-Aldrich. Goat anti-mouse Alexa 488
(1:1000), goat anti-rabbit Alexa 488 (1:1000), and goat anti-rabbit
Alexa 568 (1:1000) antibodies were purchased from Life Technologies.
Donkey anti-mouse IRDye800CW (1:20000) and donkey anti-mouse IRDye680LT
(1:20000) were bought from LI-COR Biosciences.

Burczyk M., Burkhalter M.D., Blätte T., Matysik S., Caron M.G., Barak L.S, & Philipp M. (2015). Phenotypic Regulation of the Sphingosine 1-Phosphate Receptor Miles Apart by G Protein-Coupled Receptor Kinase 2. Biochemistry, 54(3), 765-775.

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Comprehensive Immunoblotting Analysis

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Primary antibodies were rabbit anti-phospho-AKTS473, rabbit anti-AKT, rabbit anti-phospho-S6S240/244, rabbit anti-S6, rabbit anti-phospho-ERK1/2T202/204, rabbit anti-ERK1/2, rabbit anti-MAP2K4, anti-phospho-MAP2K4S257/T261, rabbit anti-p110α and rabbit anti-p85 (Cell Signaling), mouse anti-survivin, mouse anti-NEK9 and rabbit anti-β-actin (Santa Cruz). Secondary horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse antibodies were from Jackson ImmunoResearch Laboratories.

Mundt F., Rajput S., Li S., Ruggles K., Mooradian A.D., Mertins P., Gillette M.A., Krug K., Guo Z., Hoog J., Erdmann-Gilmore P., Primeau T., Huang S., Edwards D.P., Wang X., Wang X., Kawaler E., Mani D.R., Clauser K.R., Gao F., Luo J., Davies S.R., Johnson G.L., Huang K.L., Yoon C.J., Ding L., Fenyö D., Ellis M.J., Townsend R.R., Held J.M., Carr S.A, & Ma C.X. (2018). Mass spectrometry-based proteomics reveals potential roles of NEK9 and MAP2K4 in resistance to PI3K inhibitors in triple negative breast cancers. Cancer research, 78(10), 2732-2746.

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Immunoblotting Analysis of IGF-1R and IR Signaling

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To analyze IR and IGF-1R gene deletion efficiency peritoneal macrophages were enriched by plastic adhesion, cell lysates were resolved on a 4-12% reducing BisTris SDS-PAGE gel (NUPAGE, Invitrogen) and transferred to a nitrocellulose membrane (Hybond C-extra, Amersham Biosciences). Immunoreactive products were detected using the following primary antibodies: rabbit anti-IGF-1Rβ (C-20), rabbit anti-IRβ (C-19) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-α-Tubulin (Sigma-Aldrich, St. Louis, MO, USA). Phosphorylation of proteins were detected in lysates and resolved in SDS-PAGE gel as described above; primary antibodies included rabbit anti-p38α MAPK, rabbit anti-phosphop38α MAPK^T180/Y182, rabbit anti-Akt, rabbit anti-phospho-Akt^S473, rabbit anti-phospho-Akt^T308 (Cell Signaling Technology, Beverly, MA, USA), mouse anti-GAPDH (Calbiochem, La Jolla, CA, USA), mouse anti-α-Tubulin. Bound primary antibody was detected using a rabbit anti-mouse HRP-conjugated secondary antibody and a swine anti-rabbit HRP-conjugated antibody (Dako, Glostrup, Denmark). Detection of bound secondary antibody was accomplished using the enhanced chemiluminescence Western blot detection system ECL (Perkin Elmer, Waltham, MA, USA).

Knuever J., Willenborg S., Ding X., Akyüz M.D., Partridge L., Niessen C.M., Brüning J.C, & Eming S.A. (2015). Myeloid cell-restricted Insulin/IGF-1 receptor deficiency protects against skin inflammation. Journal of immunology (Baltimore, Md. : 1950), 195(11), 5296-5308.

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VEGFR Signaling Pathway Analysis

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Antibodies: goat-anti-VEGFR1 (#AF321), goat-anti-VEGFR2 (#AF357), rabbit-anti-phospho-VEGFR2-Y1214 (#AF1766) (R&D Systems, Minneapolis, MN, USA), rabbit-anti-Akt (#9272S), rabbit-anti-phospho-Akt (S473; #4060B), rabbit-anti-ERK1/2 (#9102S), mouse-anti-phospho-ERK1/2 (T202/Y204; #9106S), rabbit-anti-neuropilin 1 (NRP1; #3725S), rabbit-anti-phospho-VEGFR2-Y951 (#4991S), rabbit-anti-phospho-VEGFR2-Y1059 (#3817S), rabbit-anti-phospho-VEGFR2-Y1175 (#2478S; Cell Signaling Technology, Danvers, MA, USA), rabbit-anti-phospho-VEGFR2-Y1054 (Clone D1W; #04-894; Merck Millipore, Watford, UK), mouse-anti-α-tubulin (Clone DM1A; #T6199; Sigma Aldrich, Poole, UK), mouse-anti-PECAM-1 (CD31; #sc-65260; Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-ubiquitin (FK2; #14220; Caymen Chemical, MI, USA), mouse-anti-clathrin heavy chain antibody (X22; #ab2731; Abcam, Cambridge, UK), sheep-anti-TGN46 (#AHP500GT; AbD Serotec, Oxford, UK). Endothelial cell growth medium (ECGM) was from PromoCell (Heidelberg, Germany). Recombinant human VEGF-A₁₆₅ was from Genentech Inc. (San Francisco, CA, USA), both VEGF-A₁₂₁ and VEGF-A₁₄₅ was from Promocell.

Fearnley G.W., Smith G.A., Abdul-Zani I., Yuldasheva N., Mughal N.A., Homer-Vanniasinkam S., Kearney M.T., Zachary I.C., Tomlinson D.C., Harrison M.A., Wheatcroft S.B, & Ponnambalam S. (2016). VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis. Biology Open, 5(5), 571-583.

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