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Erk1 2 t202 y204

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ERK1/2 T202/Y204 is a laboratory reagent used to detect the phosphorylation of extracellular signal-regulated kinases 1 and 2 at threonine 202 and tyrosine 204 residues. This phosphorylation is a key event in the activation of the ERK signaling pathway.

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Lab products found in correlation

Akts473, by Cell Signaling Technology (4 mentions) Erk1 2, by Cell Signaling Technology (3 mentions) Aktt308, by Cell Signaling Technology (2 mentions) Halt protease and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions) Irdye 680rd goat anti mouse, by LI COR (1 mentions) Odyssey clx imaging system, by LI COR (1 mentions) Chemidoc touch, by Bio-Rad (1 mentions) Ecl prime, by GE Healthcare (1 mentions) Rabbit anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Abcam (1 mentions) Mouse anti tubulin, by Merck Group (1 mentions) Irdye 800cw goat anti rabbit, by LI COR (1 mentions) Criterion gel, by Bio-Rad (1 mentions) Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions) Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Rapamycin, by Cell Signaling Technology (1 mentions) Ly294002, by Cell Signaling Technology (1 mentions) S6k1t389, by Cell Signaling Technology (1 mentions) 4e bpt37 46, by Cell Signaling Technology (1 mentions) Mtors2448, by Cell Signaling Technology (1 mentions) U0126, by Cell Signaling Technology (1 mentions)

Spelling variants of this product

ERK1/2 (1487 citations) P-ERK1/2 (T202/Y204) (60 citations) Rabbit anti-phospho-ERK1/2 (T202/Y204) (4 citations) Anti–T202/Y204-phosphorylated ERK1/2 (3 citations) Anti-phospho-ERK1/2 IgG (T202/Y204; #9101) (2 citations) Anti-phospho-ERK1/2 MAPK (T202/Y204) mouse antibody (2 citations) P-ERK1/2 (T202/Y204) (9101) (2 citations) Anti-phospho-p44/42 MAPK (ERK1/2) (T202/Y204) (2 citations) Anti-phospho-ERK1/2-T202/y204 antibody (2 citations) Anti-p44/42 MAPK (Erk1/2) T202/Y204 (2 citations)

7 protocols using erk1 2 t202 y204

1

Immunoblotting Analysis of Protein Signaling

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Proteins were resolved on 10–12% SDS-PAGE for transfer to Standard PVDF, or polyvinylidene difluoride for LI-COR fluorescence imaging (PVDF/FL; EMD Millipore, Billerica, MA, USA) membranes for immunoblotting using the following antibodies: Rabbit anti-PAK1, phospho extracellular signal-related kinase (ERK1/2T202/Y204), mouse anti-ERK1/2, B cell lymphoma 2 (Bcl2), and cleaved caspase 3 (Cell Signaling, Danvers, MA, USA), phospho Bcl2S70 and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Abcam, Cambridge, MA, USA), mouse anti-tubulin (Sigma); all used at 1:1000 dilutions. IRDye 680 RD goat anti-mouse and IRDye 800CW goat anti-rabbit (LI-COR, Lincoln, NE, USA) antibodies were used at 1:15,000 dilutions, and goat anti-mouse and anti-rabbit horseradish peroxidase secondary antibodies (Bio-Rad, Irvine, CA, USA) were used at 1:5000 dilutions. All antibodies were validated for specificity based upon predicted molecular weight of immunoreactive band, selective loss of signal using siRNA or gain of signal using overexpression, and blocking peptides. Immunoreactive bands were visualised with enhanced chemiluminescence (ECL)/Prime (GE Healthcare, Buckinghamshire, UK) and imaged using a Chemi-Doc Touch (Bio-Rad). Phosphorylated and total ERK1/2 blots were imaged using the Odyssey CLx imaging system (LI-COR).
Ahn M., Yoder S.M., Wang Z., Oh E., Ramalingam L., Tunduguru R, & Thurmond D.C. (2016). The p21-activated kinase (PAK1) is involved in diet-induced beta cell mass expansion and survival in mice and human islets. Diabetologia, 59(10), 2145-2155.
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2

Immunoblotting of Insulin Signaling Proteins

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Proteins were extracted in Tissue Protein Extraction Reagent (T-PER) with the addition of Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific) using a PowerGen 700 tissue homogenizer (Fisher Scientific). Proteins (25 μg) were separated on 10% Criterion gels (Bio-Rad), transferred to nitrocellulose and probed using standard immuno-blotting techniques. Blots were incubated overnight at 4°C with primary antibodies (IGF1R(Y1135/1136)/InsR(Y1150/1151), IGF1R(Y1316), IGF1R(Y980), IRS-1(S307), PI3Kp85(Y458)/p55(Y199), PDK1(S241), Akt(T308), Akt(S473), mTOR(S2448), S6 ribosomal protein(S235/236) and p44/42 (Erk1/2)(T202/Y204) (Cell Signaling) and NR4A2 (Nurr1 M-196, Santa Cruz Biotechnology). HRP-conjugated secondary antibodies (Cell Signaling) were used and immunoreactive bands were detected with the ECL Western Blotting Chemiluminescent Substrate (Pierce). Bands were visualized using Bio-Rad XRS+ ChemiDoc station and expressed relative to corresponding non-phosphorylated protein and GAPDH expression (Cell Signaling) as previously described (Mavrommatis et al., 2013 (link)).
Solís C., Thompson W.C., Peña J.R., McDermott-Roe C., Langa P., Warren C.M., Chrzanowska M., Wolska B.M., Solaro R.J., Pieter D.e.t.o.m.b.e, & Goldspink P.H. (2022). Mechano-growth factor E-domain modulates cardiac contractile function through 14-3-3 protein interactomes. Frontiers in Physiology, 13, 1028345.
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3

Signaling Pathway Analysis Protocol

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Leflunomide and A77 1726 were kindly provided by Cinkate Corporation (Oak Park, IL). PPP was purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). U0126, LY294002, and rapamycin were purchased from Cell Signaling Technology (Danvers, MA). OSI-906 was purchased from Selleckchem.com (Houston, TX). Antibodies against ERK1/2, MEK1/2, Raf-1, p90 RSK, GSKα/β, 4E-BP, PDK1, AKT, mTOR, S6K1, and S6 and their corresponding phosphor antibodies including ERK1/2T202/Y204, MEK1/2S217/S221, Raf-1S338, p90 RSKT356/S360, GSKα/βS21/9, 4E-BPT37/46, PDKS241, AKTS473, AKTT308, mTORS2448, S6K1T389, S6S235/236 insulin receptor substrate-1S1101 (IRS-1S1101), and CADS1859 were purchased from Cell Signaling Technology.
Doscas M.E., Williamson A.J., Usha L., Bogachkov Y., Rao G.S., Xiao F., Wang Y., Ruby C., Kaufman H., Zhou J., Williams J.W., Li Y, & Xu X. (2014). Inhibition of p70 S6 Kinase (S6K1) Activity by A77 1726 and Its Effect on Cell Proliferation and Cell Cycle Progress. Neoplasia (New York, N.Y.), 16(10), 824-834.
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4

VEGF Signaling Pathway Analysis

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Cell lysis, SDS-PAGE, and immunoblotting were performed as previously described [21 (link)]. Cells pieces were lysed in 1% SDS (Fisher BioReagents, cat#BP2436-200) supplemented with 1x HALT protease and phosphatase inhibitor (ThermoFisher, cat#78441). The following antibodies were purchased from Cell Signaling Technology: VEGFR2 (cat#2479), VEGFR2 Y1175 (cat#2478), ERK1/2 (cat#4695), and ERK1/2 T202/Y204 (cat#4370). For protein assessment following ligand stimulation, cells were incubated with 10 ng/mL VEGF-A (R&D Systems cat#293VE) for 10 minutes prior to harvest and lysate generation.
Lowery C.D., Blosser W., Dowless M., Renschler M., Perez L.V., Stephens J., Pytowski B., Wasserstrom H., Stancato L.F, & Falcon B. (2019). Anti-VEGFR2 therapy delays growth of preclinical pediatric tumor models and enhances anti-tumor activity of chemotherapy. Oncotarget, 10(53), 5523-5533.
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5

Immunoblotting of Apoptotic and Signaling Proteins

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Cells were lysed and immunoblotting was performed as previously described15 (link),16 (link) using antibodies against cleaved caspase-3 (Asp175), actin, phospho-EGFR-Y1068, EGFR, phospho-protein kinase B (AKT)-S473, pan-AKT, phospho-signal transducer and activator of transcription (STAT)3-Y705, STAT3, phospho-extracellular signal-regulated kinase (ERK)1/2-T202/Y204, and ERK1/2 (Cell Signaling Technology, St Quentin en Yvelines, France), PARP (SantaCruz Biotechnology, Heidelberg, Germany), acetylated α-tubulin and α-tubulin (Abcam, Paris, France), and p21WAF1 (Merck Millipore, Molsheim, France).
Jeannot V., Busser B., Vanwonterghem L., Michallet S., Ferroudj S., Cokol M., Coll J.L., Ozturk M, & Hurbin A. (2016). Synergistic activity of vorinostat combined with gefitinib but not with sorafenib in mutant KRAS human non-small cell lung cancers and hepatocarcinoma. OncoTargets and therapy, 9, 6843-6855.
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6

Western Blotting for DNA Damage Response

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Cell lysis, SDS-PAGE, and immunoblotting were performed as previously described (10 (link)). Cells or tumor pieces were lysed in 1% SDS (Fisher BioReagents, cat#BP2436–200) supplemented with 1x HALT protease and phosphatase inhibitor (ThermoFisher, cat#78441). The following antibodies were purchased from Cell Signaling: CHK1 (cat#2360), CHK1 S296 (cat#90178), CHK1 S345 (cat#234), WEE1 (cat#4936), WEE1 S642 (cat#4910), PARP (cat#9542), AKT (cat#9272), AKT S473 (cat#4060), ERK1/2 (cat#4695), ERK1/2 T202/Y204 (cat#4370), MEK1/2 (cat#4694), MEK1/2 S217/221 (cat#9154), and BCL-xL (cat#2764). Additional antibodies included CHK2 (StressGen, cat#KAM-CC112), RPA32/2 (AbCam, cat#ab61184), RPA32/2 S4/8 (Bethyl, cat#A300–245), γH2AX (Millipore, cat#05–636), and GAPDH (Millipore, cat#MAB374).
Lowery C.D., Dowless M., Renschler M., Blosser W., VanWye A.B., Stephens J.R., Iversen P.W., Lin A.B., Beckmann R.P., Krytska K., Cole K.A., Maris J.M., Hawkins D.S., Rubin B.P., Kurmasheva R.T., Houghton P.J., Gorlick R., Kolb E.A., Kang M.H., Reynolds C.P., Erickson S.W., Teicher B.A., Smith M.A, & Stancato L.F. (2018). Broad spectrum activity of the checkpoint kinase 1 inhibitor prexasertib as a single agent or chemopotentiator across a range of preclinical pediatric tumor models. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(7), 2278-2289.
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7

Signaling Pathways in Platelet Activation

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Cross-linked collagen-related peptide (CRP-XL) was from R. Farndale (Department of Biochemistry, University of Cambridge, UK). Fibrillar HORM collagen (type I) derived from equine tendon from Takeda (Linz, Austria). Recombinant murine thrombopoietin (TPO) was from PeproTech (London, UK). D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone (PPACK) was from Calbiochem (Merck Chemicals, Watford, UK). 3,3'-Dihexyloxacarbocyanine iodide (DiOC6) and TxB2 ELISA kit were from Enzo Life Sciences (Exeter, UK). CHRONO-LUME® reagent was from CHRONO-LOG (Labmedics, Abingdon, UK). Akt S473 (#4060), Akt T308 (#13038), Akt (#9272), ERK1/2 T202/Y204 (#9101), GSK-3α/β S21/9 (#9331), JAK2 Y1007/1008 (#3771), JAK2 (#3230), p110α (#4249), Phospho-(Ser) PKC Substrate (#2261), STAT5/ Y694 (#4322), and STAT5/ (#9358) were from Cell Signaling Technology (New England Biolabs, Hitchin, UK). GAPDH (#sc-25778) antibody was from Santa Cruz Biotechnology (Insight Biotechnology, Middlesex, UK). PD184352, VX-702 and TGX-221 were from Bio-Techne (Abingdon, UK). PIK-75 and A66 were from Cayman Chemicals (Cambridge Bioscience, Cambridge, UK). Secondary antibodies for immunoblotting were from Jackson Immunoresearch (Stratech Scientific, Ely, UK). All other reagents were from Sigma (Poole, UK), unless otherwise indicated.
Blair T.A., Moore S.F., Walsh T.G., Hutchinson J.L., Durrant T.N., Anderson K.E., Poole A.W, & Hers I. (2018). Phosphoinositide 3-kinase p110α negatively regulates thrombopoietin-mediated platelet activation and thrombus formation. Cellular signalling, 50.
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