Phospho erk t202 y204

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-ERK (T202/Y204) is a lab equipment product that detects the dual phosphorylation of extracellular signal-regulated kinase (ERK) at threonine 202 and tyrosine 204 residues. This phosphorylation is a key activation event in the mitogen-activated protein kinase (MAPK) signaling pathway.

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Lab products found in correlation

Phospho akt s473, by Cell Signaling Technology (5 mentions) Total erk, by Cell Signaling Technology (3 mentions) Anti syk clone 5f5, by BioLegend (2 mentions) Anti lyn clone lyn 01, by BioLegend (2 mentions) Anti ly6g clone rc6 8c5, by R&D Systems (2 mentions) Cleaved caspase 3, by Cell Signaling Technology (2 mentions) Gefitinib, by Selleck Chemicals (2 mentions) β actin, by Cell Signaling Technology (2 mentions) Gf109203x gfx, by Merck Group (2 mentions) Stem cell factor (scf), by Thermo Fisher Scientific (2 mentions) Phospho p38t180 y182, by Cell Signaling Technology (2 mentions) Xtt proliferation assay kit, by Bio-Techne (2 mentions) Total p38, by Cell Signaling Technology (2 mentions) Phospho pkcβ s660, by Cell Signaling Technology (2 mentions) Phospho pkcα s657 y658, by Abcam (2 mentions) Total pkcα, by Abcam (2 mentions) Total pkcβ, by Santa Cruz Biotechnology (2 mentions) Pe conjugated rat anti mouse cd117 kit, by BioLegend (2 mentions) Fitc conjugated anti mouse fcεr1 antibodies, by BioLegend (2 mentions) Brdu proliferation assay kit calbiochem, by Merck Group (2 mentions)

Close spelling variants of this product

Phospho-ERK (527 citations) ERK/phospho-ERK (3 citations) Anti-phospho-ERK (T202/Y204) (3 citations) Phospho-p44/42 MAPK (Erk 1/2) T202/Y204 (2 citations)

18 protocols using phospho erk t202 y204

Capillary Electrophoresis for DIPG Protein

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Capillary electrophoresis was conducted on protein lysates from the DIPG autopsy specimens using the automated Wes system (Protein Simple) with the 12- to 230-kDa Separation module (SM-W004) and the anti-rabbit detection module (DM-001) following the manufacturer's instructions and analyzed with Compass software. The following primary antibodies were used: ERK1/2 (1:100, Cell Signaling Technology, 9102), phospho-ERK^T202/Y204 1:100 (Cell Signaling Technology, 9101), and α-actin (1:200, Cell Signaling Technology, 6487). Goat anti-rabbit HRP conjugate (ProteinSimple, 042–206) was used as a secondary antibody.

Carvalho D.M., Richardson P.J., Olaciregui N., Stankunaite R., Lavarino C., Molinari V., Corley E.A., Smith D.P., Ruddle R., Donovan A., Pal A., Raynaud F.I., Temelso S., Mackay A., Overington J.P., Phelan A., Sheppard D., Mackinnon A., Zebian B., Al-Sarraj S., Merve A., Pryce J., Grill J., Hubank M., Cruz O., Morales La Madrid A., Mueller S., Carcaboso A.M., Carceller F, & Jones C. (2021). Repurposing Vandetanib plus Everolimus for the Treatment of ACVR1-Mutant Diffuse Intrinsic Pontine Glioma. Cancer Discovery, 12(2), 416-431.

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Multiparameter Phosphoprotein Analysis

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Abs directed against phosphotyrosine (PY1000), phospho–spleen tyrosine kinase (Syk) (Y525/526), phospho-pan Src (Y527 and Y416), phospho-Akt (S473), phospho-p38 (T180, Y182), and phospho-Erk (T202, Y204) were from Cell Signaling Technology. Anti-Syk (clone 5F5) and anti-Lyn (clone LYN-01) were from BioLegend. Anti-Ly6G (clone RC6-8C5) was obtained from R&D Systems. Anti-BSA and anti-lactoferrin Abs were from Sigma and an Ab against β-COP was a gift from Nick Ktistakis (The Babraham Institute, Cambridge, U.K.). HRP-conjugated secondary Abs were from Santa Cruz Biotechnology and Bio-Rad. Fluorescently conjugated Abs for flow cytometry were obtained from eBioscience (F4/80, GR1), BioLegend (CD11b, CD11a, CD16/32, Ly6G, CD62L, CD19), and BD (Ly6C).

Vermeren S., Miles K., Chu J.Y., Salter D., Zamoyska R, & Gray M. (2016). PTPN22 Is a Critical Regulator of Fcγ Receptor–Mediated Neutrophil Activation. The Journal of Immunology Author Choice, 197(12), 4771-4779.

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Comprehensive Protein Extraction and Analysis

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Total proteins were extracted through 30-minute incubation on ice with Nonidet P-40 lysis buffer containing protease and phosphatase inhibitors, and resolved on Bolt Bis-Tris Plus polyacrylamide gels (Life Technologies). Antibodies against PARP (cat# 9532S), Phospho-Akt (S473; cat# 4060S), Akt (cat# 4685S), Phospho-Erk (T202/Y204; cat# 9101S), Erk1/2 (cat# 9102S), Phospho-S6 ribosomal protein (S235/236; cat# 2211S), total S6 ribosomal protein (cat# 2317S), Phospho-S6 kinase (cat# 9234S), total S6-kinase (cat# 2708S), Tuberin/TSC2 (cat# 4308S), Phospho-RSK (S380) (cat# 9335), total RSK (cat# 9355), Fatty Acid Synthase (cat# 3180S), Acetyl-CoA Carboxylase (cat# 3676S), Stearoyl-CoA desaturase 1 (cat# 2794S), CCTα (cat# 6931S) and BrdU (5292S) were obtained from Cell Signaling Technology (Danvers, MA). Anti-beta actin antibody (cat# A5316) was obtained from Millipore Sigma (St. Louis, MO) and anti-CPT1A antibody (cat# ab128568) from Abcam (Cambridge, MA).

Feng Y., Mischler W.J., Gurung A.C., Kavanagh T.R., Androsov G., Sadow P.M., Herbert Z.T, & Priolo C. (2020). Therapeutic targeting of the secreted lysophospholipase D autotaxin suppresses tuberous sclerosis complex-associated tumorigenesis. Cancer research, 80(13), 2751-2763.

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Antibody and Protein Assay Protocol

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Anti-MSI2 (#ab76148), anti-MSI1 (#ab21628), and anti-β-actin HRP conjugated (#ab49900) antibodies and recombinant MSI2 protein (#ab167853) were obtained from Abcam, (Cambridge, UK). Anti-EGF receptor (#4267), phospho-EGFR (Y1068) (mAb #2234), HER3/ErbB3 (#12708), HER2/ErbB2 (#4290), Smad3 (#9523), phospho-AKT (T308) (#13038), total AKT (#2920), phospho-ERK (T202/Y204) (#4370), total ERK (#4696), phospho-p70S6K (T389) (#9234), total p70S6K (#2708), and normal Rabbit IgG (#2729) were obtained from Cell Signaling, (Danvers, MA). Erlotinib and afatinib were obtained from LC Laboratories (Woburn, MA), doxycycline from Sigma-Aldrich (D9891, Darmstadt, Germany). SUPERase-In RNAse inhibitor was obtained from Thermo Fisher Scientific, (AM2694 Waltham, MA). Osimertinib was obtained from Selleckchem (#S7297, Houston, TX)

Makhov P., Bychkov I., Faezov B., Deneka A., Kudinov A., Nicolas E., Brebion R., Avril E., Cai K.Q., Kharin L.V., Voloshin M., Frantsiyants E., Karnaukhov N., Kit O.I., Topchu I., Fazliyeva R., Nikonova A.S., Serebriiskii I.G., Borghaei H., Edelman M., Dulaimi E., Golemis E.A, & Boumber Y. (2021). Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC). Oncogenesis, 10(3), 29.

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SHP2, PD-L1, and ERK Western Blot Analysis

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Cell lysates were extracted from the samples harvested at 18 hpi. The lysates were mixed with a sample buffer (Solarbio, Beijing, China) followed by heat treatment at 95 °C for 5 min. The samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Bands were detected with primary antibodies specific to SHP2 (3397T, Cell Signaling Technology, Danvers, MA, USA), phospho-SHP2 (Y542) (3751T, Cell Signaling Technology, Cambridge, MA, USA), PD-L1 (3751T, Cell Signaling Technology), ERK (4695T, Cell Signaling Technology, MA, USA), phospho-ERK (T202/Y204) (4370T, Cell Signaling Technology), β-actin (R019, Transgen biotech, China), and anti-H1N1-NP (generated in our laboratory) at 4 °C overnight prior to incubation with horseradish peroxidase-conjugated goat anti-mouse (125229, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) or anti-rabbit IgG (131879, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) incubation for 2 h. The blots were developed using the FluorChem M Imaging System (Protein Simple, San Jose, CA, USA). β-actin was used as a reference of internal standard.

Ning H., Chiu S.H., Xu X., Ma Y., Chen J.L, & Yang G. (2023). The Immunosuppressive Roles of PD-L1 during Influenza A Virus Infection. International Journal of Molecular Sciences, 24(10), 8586.

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Antibodies for Signaling Pathway Analysis

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Antibodies directed against phosphotyrosine (PY1000), phospho-Syk (Y525/526), phospho-pan Src (Y527 and Y416), phospho-Akt (S473), phospho-p38 (T180, Y182) and phospho-Erk (T202, Y204) were from Cell Signaling Technology. Anti-Syk (clone 5F5) and anti-Lyn (clone LYN-01) were from Biolegend. Anti-Ly6G (clone RC6-8C5) was obtained from R&D Systems. Anti-BSA and anti-lactoferrin antibodies were from Sigma and an antibody against β-COP was a gift from Nick Ktistakis (The Babraham Institute, Cambridge). HRP-conjugated secondary antibodies were from Santa Cruz Biotechnology and Biorad. Fluorescently-conjugated antibodies for flow cytometry were obtained from eBioscience (F4/80, GR1), Biolegend (CD11b, CD11a, CD16/32, Ly6G, CD62L, CD19) and BD (Ly6C).

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Protein Extraction and Western Blot Analysis

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After treatments, slices were collected in dry ice and homogenized in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton) containing protease and phosphatase inhibitor cocktail (Thermo). Protein concentrations were measured using the BCA protein assay kit (Thermo). Equal amounts of proteins (20–30 μg) were processed for SDS-PAGE and western blot, as previously described63 . The primary antibodies used were PTEN (1:1000, Cell signaling), phospho-ERK T202/Y204 (1:2000, Cell Signaling), ERK (1:3000, Cell signaling), phospho-Akt S473 (1:1000, Cell signaling), Akt (1:2000, Cell signaling), phospho-S6 S240/S244 and S6 (both 1:1000, Cell signaling), phospho-eIF2α S51 (1:500, Cell signaling), eIF2α (1:1000, Cell signaling), PP2A B56 alpha subunit (1:200, Santa Cruz), GLUA1 N-terminal (1:200, Santa Cruz), GLUA1 C-terminal (1:1000, Millipore), α-spectrin (1:2000, Millipore), PHLPP1 (1:1000, Millipore), actin (1:10000, Millipore).

Zhu G., Briz V., Seinfeld J., Liu Y., Bi X, & Baudry M. (2017). Calpain-1 deletion impairs mGluR-dependent LTD and fear memory extinction. Scientific Reports, 7, 42788.

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Profiling Kinase and Cell Signaling Pathways

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Protease and phosphatase inhibitor cocktails were purchased from Roche and CHAPS was obtained from Pierce. Gefitinib and Erlotinib were from Selleckchem. The IKKβ inhibitor, CmpdA, was a gift from Dr. Albert Baldwin at the University of North Carolina (Chapel Hill, NC, USA).
Antibodies against phospho-EGFR-Y1068 (CST-3733), phospho-HER2-Y1248 (CST-2247), HER2 (CST-4290), phospho-HER3-Y1289 (CST-2842), HER3 (CST-12708), phospho-p65-S536 (CST-3033), p65 (CST-6956), phospho-IKKα S176/β S177 (CST-2697 and CST-2078), IKKα (CST-2682), IKKβ (CST-8943), phospho-Akt-S473 (CST-4508), Akt (CST-2938), phospho-S6K-T389 (CST-9205), S6K (CST-9202), phospho-ERK-T202/Y204 (CST-4370), ERK (CST-4348), cleaved caspase-3 (CST-9664) and GAPDH (CST-5174) were purchased from Cell Signalling. Anti-EGFR (SC-03) came from Santa Cruz Biotechnology, along with horse radish peroxidase-labelled anti-mouse and anti-rabbit secondary antibodies.

Li Z., Liao J., Yang Z., Choi E.Y., Lapidus R.G., Liu X., Cullen K.J, & Dan H. (2018). Co-targeting EGFR and IKKβ/NF-κB signalling pathways in head and neck squamous cell carcinoma: a potential novel therapy for head and neck squamous cell cancer. British Journal of Cancer, 120(3), 306-316.

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Immunoblotting Analysis of Cellular Signaling

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Resistant cells were removed from drug selection 72 hours before immunoblotting experiments. Cells were washed on ice with cold PBS and lysed in radioimmunoprecipitation (RIPA) buffer (250mM Tris-HCl pH 7.5, 75mM NaCl, 1% NP-40/IGEPAL, 0.1% sodium dodecyl sulfate) supplemented with complete protease inhibitor cocktail (Roche), 40mM sodium fluoride, 1mM sodium orthovanadate, and 1uM okadaic acid. Lysates were subjected to SDS-PAGE (4-12% gels) followed by immunoblotting with the indicated antibodies and detection by Western Lightning ECL reagent (Perkin-Elmer). The following antibodies were obtained from Cell Signaling Technologies: phospho-ERK (T202/Y204; 1:1000; # 9101), ERK (1:1000, #9102), phospho-AKT (S473; 1:500; #9271), AKT (1:1000; #9272), phospho-S6 (S240/244; 1:1000; #2215), S6 (1:1000; #2217), BIM (1:1000; #2819), HRP-conjugated anti-mouse (1:3000; #7076), and HRP-conjugated anti-rabbit (1:3000; #7074). Phospho-EGFR antibody was obtained from Abcam (Y1068; 1:1000; #EP774Y), EGFR from BD Transduction Laboratories (1:500; #610017), and actin from Sigma-Aldrich (1:3000; #A2066). Phospho-RTK arrays were purchased from R&D Systems (#ARY001B), and assays were run on cells maintained in 10% FBS using the manufacturer’s protocol.

Meador C.B., Jin H., de Stanchina E., Nebhan C.A., Pirazzoli V., Wang L., Lu P., Vuong H., Hutchinson K.E., Jia P., Chen X., Eisenberg R., Ladanyi M., Politi K., Zhao Z., Lovly C.M., Cross D.A, & Pao W. (2014). Optimizing the sequence of anti-EGFR targeted therapy in EGFR-mutant lung cancer. Molecular cancer therapeutics, 14(2), 542-552.

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Immunoblotting of Sciatic Nerve Proteins

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Immunoblotting was performed as described in [32 (link)]. Separate pooled protein lysates were prepared from both intact and crushed sciatic nerves. The following primary antibodies were used: phospho-c-Jun (Ser73, 1:1000, Cell Signaling, USA, 8752), Neuregulin 1 (1:250, Santa Cruz, USA, clone C-20), Erk (1:500, Cell Signaling, USA), phospho-Erk (T202/Y204, 1:500, Cell Signaling, USA), ErbB2 (1:500, Cell Signaling, USA), GAPDH (1:1000, Santa Cruz, USA, 6C5) and merlin (1:500, Santa Cruz, USA A19). Results were quantified using gel analysis software by ImageJ. Density values were normalized to GAPDH and appropriate controls of transfection or wild-type tissue, respectively. In the case of phospho-specific detection of proteins, their acquired densities were referred to signals derived from related pan-antibodies that served as loading control (e.g., phospho-Erk to Erk signals).

Schulz A., Büttner R., Hagel C., Baader S.L., Kluwe L., Salamon J., Mautner V.F., Mindos T., Parkinson D.B., Gehlhausen J.R., Clapp D.W, & Morrison H. (2016). The importance of nerve microenvironment for schwannoma development. Acta Neuropathologica, 132, 289-307.

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