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P473AKT is a rabbit monoclonal antibody that specifically recognizes AKT protein phosphorylated at serine 473. It is suitable for use in Western blotting and immunohistochemistry applications to detect the phosphorylation state of AKT.

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Lab products found in correlation

P 308 akt, by Cell Signaling Technology (3 mentions) 4e bp1, by Cell Signaling Technology (3 mentions) Ecl plus developing system, by Cytiva (2 mentions) Deptor, by Novus Biologicals (2 mentions) Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (2 mentions) β actin, by Cell Signaling Technology (2 mentions) Vinculin, by Merck Group (2 mentions) Antibody to vinculin, by Merck Group (1 mentions) Total akt, by Cell Signaling Technology (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) P p70s6k, by Cell Signaling Technology (1 mentions) Anti au1 antibody, by Biosynth (1 mentions) P gsk3a b, by Cell Signaling Technology (1 mentions) P mdm2, by Cell Signaling Technology (1 mentions) Mlc2v, by Synaptic Systems (1 mentions) T akt, by Cell Signaling Technology (1 mentions) T p70s6k, by Cell Signaling Technology (1 mentions) Gapdh, by Biosynth (1 mentions) Caspase 1 p20, by Genentech (1 mentions) β tubulin sc 9104, by Cell Signaling Technology (1 mentions)

8 protocols using p473akt

1

Skeletal Muscle Signaling Pathway Analysis

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RT-PCR and immunoblotting were conducted as previously described.41 (link) Skeletal muscle underwent evaluation of Akt and AMPK activation using antibodies obtained from Cell Signaling Technology (Danvers, MA): p-308-Akt (#4056), p-473-Akt (#4058), total Akt (# 9272), p-172-AMPK (#2535), and GAPDH (# 51332S). Antibody to vinculin was obtained from Sigma (St. Louis, MO; SAB4200080).
Gao M.H., Giamouridis D., Lai N.C., Guo T., Xia B., Kim Y.C., Huu V.A., Skowronska-Krawczyk D., Lantier L., Bhargava R, & Hammond H.K. (2019). Urocortin 2 Gene Transfer Improves Glycemic Control and Reduces Retinopathy and Mortality in Murine Insulin Deficiency. Molecular Therapy. Methods & Clinical Development, 17, 220-233.
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2

Western Blot Analysis of Neural Markers

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Cell lysates were created and Western blotting conducted as described (24 (link)), blocking without detergent. Membranes were probed with antibodies for MAF (Imgenex), SOX9 (Santa Cruz), S100β (Dako), MBP (Chemicon), BLBP (Millipore), DEPTOR (Novus), cleaved-caspase 3, pS6, S6, p4E-BP1, 4E-BP1, p473AKT, AKT, and β-ACTIN (Cell Signaling) as a loading control. Horseradish peroxidase-conjugated secondary antibodies (BioRad) were used with ECL Plus developing system (Amersham Biosciences).
Brundage M.E., Tandon P., Eaves D.W., Williams J.P., Miller S.J., Hennigan R.H., Jegga A., Cripe T.P, & Ratner N. (2014). MAF mediates crosstalk between Ras-MAPK and mTOR signaling in NF1. Oncogene, 33(49), 5626-5636.
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3

Adenylyl Cyclase Protein Expression Analysis

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Expression of adenylyl cyclase related proteins were analyzed using
Agilent Gene Expression Microarray technique by PhalanxBio Inc. (CA, San Diego).
C1C2 protein was detected by anti-AU1 antibody (Fitzgerald, 1:2,000) or
anti-AC5/6 antibody (Santa Cruz, 1:200). Additional antibodies included: GAPDH
(Fitzgerald, 1:20,000); cMLCK (Abgent, 1:1,000); MLC2v (Synaptic Systems,
1:1,000) PKA catalytic subunit (BD Transduction, 1:1,000); p-ERK1/2, p38,
p-PKARII α and β (Santa Cruz Biotech, 1:200); PLB (Affinity
Bioreagents, 1:5,000); phospho-16-PLB (Badrilla, 1:3,000); S100A1 (Epiyomics,
1:1,000); SERCA2a antibody (Enzo, 1:1,000); phospho-αB-crystallin
(CryAB) and total CryAB antibodies (Enzo, 1:1,000); P-308-Akt, P-473-Akt, T-Akt,
p-GSK3a/b, p-MDM2, P-p70S6K, T-p70S6K, and phospho-S22/23 troponin I (TnI) (Cell
Signaling, 1:1,000 each); Vinculin (Sigma, 1:100,1000).
Gao M.H., Lai N.C., Giamouridis D., Kim Y.C., Tan Z., Guo T., Dillmann W.H., Suarez J, & Hammond H.K. (2016). Cardiac-Directed Expression of Adenylyl Cyclase Catalytic Domain Reverses Cardiac Dysfunction Caused by Sustained Beta-Adrenergic Receptor Stimulation. JACC: Basic to Translational Science, 1(7), 617-629.
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4

Immunoblotting of Apoptosis Regulators

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Tissues were lysed in buffer B150 (20 mM Tris-HCl pH 8.0, 150 mM KCl,
10% glycerol, 5 mM MgCl2, and 0.1% NP40) supplemented with
Complete-mini protease inhibitors (Roche Applied Science, Cat#
11836153001) and phosphatase inhibitors (Sigma Cat# S7920, 71768,
G6376). Protein lysates were separated on SDS-PAGE and transferred to
nitrocellulose membrane. Blots were probed with antibodies against Caspase-1 p20
(Genentech), Caspase-12 (Sigma, Cat# C7611), Caspase-11 (Sigma,
Cat# C1354), β-actin (Sigma, Cat# A1978), β
-tubulin (sc-9104), AKT (Cell signalling #4691), and P473 AKT (Cell
signalling #4060). Densitometry was performed using ImageJ (NIH).
Skeldon A.M., Morizot A., Douglas T., Santoro N., Kursawe R., Kozlitina J., Caprio S., Mehal W.Z, & Saleh M. (2015). Caspase-12, but not caspase-11, inhibits obesity and insulin resistance. Journal of immunology (Baltimore, Md. : 1950), 196(1), 437-447.
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5

Ibrutinib Inhibition of BCR Signaling

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CLL cells purified through negative column selection as detailed above, were plated at 5×106 cells/500μl RPMI1640 medium without FCS and rested for 1 hour at 37°C and subsequently pre-incubated for 1 hour at 37°C as indicated with ibrutinib at 0 μM (DMSO only) or 0.25 μM, 0.5 μM or 1 μM. Cells were stimulated with goat anti-human IgM (Southern Biotech, cat #2020-08) at 10μg/ml for 15′ and subsequently pelleted through centrifugation at 4°C. Cell pellets were lyzed in lysis buffer containing 1% NP-40 detergent (#DSC41010; Dot Scientific), 150mM NACL, 25mM Tris pH 8.0 (#T6066; Sigma Aldrich), 20mM NAF, 2mM EGTA, 2mM EDTA (#ED2SS; Sigma Aldrich), supplemented with protease inhibitors (#P8340) and phosphatase inhibitors (#P0044), and sodium orthovanadate (#450243; Sigma-Aldrich, St. Louis, MO), and PMSF (#36978; Thermo Scientific). The detergent-soluble fraction of the cell lysates was cleared by centrifugation at 14,000 rpm for 10 min. Protein was fractionated through SDS-PAGE and prepared for immunoblotting using standard procedures. For immunoblotting the following antibodies were used (all rabbit anti-human antibodies from Cell Signaling Technologies): PLCy2: #3872; p1217-PLCy2: #3871; BTK: #8547; p223-BTK: #5082s; AKT: #9272; p473-AKT: #9271s; ERK: #4695; p202/204-ERK: #4370.
Amin N.A., Balasubramanian S., Saiya-Cork K., Shedden K., Hu N, & Malek S.N. (2016). Cell-intrinsic determinants of ibrutinib-induced apoptosis in Chronic Lymphocytic Leukemia. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(4), 1049-1059.
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6

Comprehensive Immunohistochemistry Protein Profiling

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Vinculin (Sigma #V4505), ER antibodies: Santa Cruz cat# sc543 or Millipore #04-820), ER pS118 (Cell Signalling #2511), ER for IHC (Ventana #790-4324 [SP1]), p308-AKT (Cell Signaling #2965), p473-AKT (Cell Signaling #4060), AKT (Cell Signaling #4691), p240/244 S6 (Cell signaling #5364), p65-4EBP1 (Cell Signaling #9456), 4EBP1 (Cell Signaling #9452), cyclinD1 (Cell Signaling #2926), Rabbit IgG (Sigma, I5006). IHC: PR (1E2) Her2 (4B5) (Ventana Discovery XT). Biotinylated secondary antibodies (Vector Labs PK-6101).
Ladd B., Mazzola A.M., Bihani T., Lai Z., Bradford J., Collins M., Barry E., Goeppert A.U., Weir H.M., Hearne K., Renshaw J.G., Mohseni M., Hurt E., Jalla S., Bao H., Hollingsworth R., Reimer C., Zinda M., Fawell S, & D'Cruz C.M. (2016). Effective combination therapies in preclinical endocrine resistant breast cancer models harboring ER mutations. Oncotarget, 7(34), 54120-54136.
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7

Western Blot Analysis of Neural Markers

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Cell lysates were created and Western blotting conducted as described (24 (link)), blocking without detergent. Membranes were probed with antibodies for MAF (Imgenex), SOX9 (Santa Cruz), S100β (Dako), MBP (Chemicon), BLBP (Millipore), DEPTOR (Novus), cleaved-caspase 3, pS6, S6, p4E-BP1, 4E-BP1, p473AKT, AKT, and β-ACTIN (Cell Signaling) as a loading control. Horseradish peroxidase-conjugated secondary antibodies (BioRad) were used with ECL Plus developing system (Amersham Biosciences).
Brundage M.E., Tandon P., Eaves D.W., Williams J.P., Miller S.J., Hennigan R.H., Jegga A., Cripe T.P, & Ratner N. (2014). MAF mediates crosstalk between Ras-MAPK and mTOR signaling in NF1. Oncogene, 33(49), 5626-5636.
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8

Immunofluorescence Characterization of Cell-Matrix Adhesions

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Cultures were fixed with 4% paraformaldehyde (2D: 20 minutes, room temperature; 3D: overnight, 4°C) and staining was performed as described (1 (link)). Primary antibodies against vinculin (hVIN-1, Sigma Aldrich; 700062, Invitrogen; V284, Santa Cruz Biotechnology), β1 integrin (AIIB2, isolated from rat hybridoma), β4 integrin (3E1, Millipore MAB1964), p397FAK (44625G,Invitrogen), p473Akt (Cell Signaling 9271S), Akt(pan)-Alexa488 (Cell Signaling C67E7), E-cadherin (610181; BD Transduction),β-catenin (610153, BD), pan-laminin (L9393, Sigma), laminin 5 (P3H9, isolated from mouse hybridoma), α6 integrin (GoH3, eBiosciences), phospho-myosin light chain kinase 2 - Thr18/Ser19 (3674, Cell Signaling), talin (T3287, Sigma), zyxin (BD, 610521), and AlexaFluor phalloidin (633-conjugate, Invitrogen) were used. Secondary antibodies used include AlexaFluor goat anti-mouse, anti-rabbit, and anti-rat (488, 568 and 633 conjugates).
Rubashkin M., Cassereau L., Bainer R., DuFort C., Yui Y., Ou G., Paszek M., Davidson M., Chen Y, & Weaver V. (2014). Force engages vinculin and promotes tumor progression by enhancing PI3-kinase activation of phosphatidylinositol (3,4,5)-triphosphate. Cancer research, 74(17), 4597-4611.
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