P erk1 2 t202 y204

Manufactured by Cell Signaling Technology

Sourced in United States

P-ERK1/2 (T202/Y204) is a lab equipment product that detects the phosphorylation of ERK1/2 at threonine 202 and tyrosine 204 residues. This phosphorylation is an important signaling event in the activation of the ERK pathway.

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Lab products found in correlation

Close spelling variants of this product

P-ERK1/2 (T202/Y204) (9101) (2 citations) Anti-p-ERK1/2 (T202/Y204), (11 citations) Anti-p-ERK1/2 (T202/Y204) (4370), (3 citations) ERK1/2 T202/Y204 (7 citations) P-ERK1/2 (1094 citations) ERK1/2 (1487 citations)

60 protocols using p erk1 2 t202 y204

Immunoblotting for Insulin Signaling Pathway

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Specific antibodies for pAkt(Ser473)(#4060, 1:5000), pAkt(Thr308)(#4056, 1:2500), Akt(#4691, 1:5000), pINSR(#3024, 1:5000), INSRβ(#3025, 1:5000), p-p70-S6K(#97596, 1:2000), S6K(#9202, 1:5000), pFoxO1(S256)(#9461, 1:2000), FoxO1(#2880, 1:5000), pGSK3β(#5558, 1:5000), GSK3α/β(#5676, 1:5000), p-ERK1/2(T202/Y204)(#9101, 1:5000), ERK1/2(#4695, 1:5000), and Hsp70(#4872, 1:2000) were obtained from (Cell Signaling Technology Inc., Danvers, MA, USA). pIRS1(#09432, 1:2500) and IRS1(#06248, 1:2500) were acquired from (Merck KGaA, Darmstadt, Germany). β-Actin(#A228, 1:5000) was procured from (Sigma-Aldrich). Peroxidase-conjugated secondary antibodies Anti-Rabbit IgG (#NA934, 1:5000) and Anti-Mouse IgG (#NA931, 1:5000) were purchased from (GE Healthcare BioSciences AB, Chicago, IL, USA). Anti-Rabbit IgG Secondary Antibody (Alexa Fluor™ 568, 1:5000) was obtained from (Thermo Fisher Scientific, Waltham, MA, USA).

Dong Q., Endo M., Kawamura G, & Ozawa T. (2022). Systematic Interrogation of the Temperature Perturbation in the Insulin Signaling Pathway for Optogenetic Stimulation. Cells, 11(19), 3136.

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Western Blot Analysis of Signaling Proteins

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Cells were lysed in RIPA buffer complemented with Set I and Set II phosphatase inhibitors at 1× (Calbiochem), and protease inhibitors at 1× (Roche). Whole cell lysate concentration was determined with Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad). Proteins were resolved on SDS-PAGE gels and electrotransferred to nitrocellulose membranes, 0.2 µm (Bio-Rad). Primary antibodies pS6_S235/236, S6, pAKT_S473, AKT, p4E-BP1_T37/46, 4E-BP1, Cleaved PARP, pAkt_T308, p62, Rictor, Raptor, HIF-1α, HIF-2α, pERK1/2_T202/Y204 (mouse), ERK, p-p90RSK_S380, RSK1/2/3, pBAD_S112, pBAD_S136, pEGFR_Y1068, cleaved-caspase3 were from Cell Signaling Technologies. VHL (Santa Cruz #FL-181). mTOR primary antibody was from Millipore. Primary antibody dilutions were to manufactures' specifications (See Table S1). Tubulin (Sigma #T5168), KU-80 (GeneTex #GTX70485) and Actin-HRP (Santa Cruz #C-11) primary antibodies served as loading controls (LC) where noted. Secondary anti-Rabbit and ant-mouse antibodies were from (Fisher) and diluted in 5% milk, 1× TBS-T solution. ECL Western Blotting Detection reagents (GE Healthcare) were used for developing blots onto autoradiography film. For difficult to detect proteins SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) was used in combination with ECL.

Bailey S.T., Zhou B., Damrauer J.S., Krishnan B., Wilson H.L., Smith A.M., Li M., Yeh J.J, & Kim W.Y. (2014). mTOR Inhibition Induces Compensatory, Therapeutically Targetable MEK Activation in Renal Cell Carcinoma. PLoS ONE, 9(9), e104413.

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Screening of RET Kinase Inhibitors

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Chemicals were from Sigma-Aldrich unless otherwise stated. Compound C3 (GW440139A), originally provided by GSK (UK), was re-synthesised at the ARUK Drug Discovery Institute at UCL [28 (link)]. LOXO-292 was purchased from MedChemExpress (USA). Recombinant human GDNF and BDNF were purchased from PeproTech (UK), and GFRα1 was purchased from R&D Systems (USA). RET shRNA constructs were purchased from Genecopoeia in psi-LVRU6GP plasmids with eGreen Fluorescent Protein (GFP) reporter genes and puromycin stable selection markers (MSH025822-LVRU6GP, USA). Antibodies used in immunofluorescence (IF) and western blot (WB) experiments were as follows: RET (C31B4, WB 1:1000, IF 1:250), RET pY905 (3221 1:1000), ERK1/2 (9102 1:1000), pERK1/2 T202/Y204 (9101 1:1000), AKT (9272 1:1000), pAKT S473 (4060 1:1000, Cell Signalling Technology (UK); GAPDH (mab374 1:5000, Merck Millipore, Germany); βIII-tubulin (IF 1:500, WB 1:3000, 302305, Synaptic Systems, Germany), GFP (GFP-1010, IF 1:1000, WB 1:5 000, Aves Labs, USA). The α-p75^NTR used in the kinase inhibitor screen was previously generated and characterised by the authors [8 (link), 29 (link), 30 (link)].

Rhymes E.R., Tosolini A.P., Fellows A.D., Mahy W., McDonald N.Q, & Schiavo G. (2022). Bimodal regulation of axonal transport by the GDNF-RET signalling axis in healthy and diseased motor neurons. Cell Death & Disease, 13(7), 584.

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Western Blot Analysis of EMT Markers

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The anti-NUAK1 antibody (22723-1-AP) and anti-β-actin antibody (23660-1-AP) were purchased from ProteinTech (Chicago, IL, USA); the anti-E-cadherin (#14472), anti-Vimentin (#46173), anti-N-cadherin antibody (#13116), TWIST1 (#90445), ZEB1 (#83243), Snail (#3879), Slug (#9585), p-c-Jun (S73) (#3270), c-Jun (#9165), p-JNK (T183/Y185) (#4668), JNK (#9252), p-Erk1/2 (T202/Y204) (#4370), Erk1/2 (#4695), p-p38 (T180/Y182) (#54470) and p38 (#9212) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). SP600125 (HY-12,041), JNK-IN-8 (HY-13,319) and puromycin dihydrochloride (HY-B1743A) were purchased from MCE (NJ, USA).

Yang H., Wei Z., Song Y., Du K., Yin N., Lu H., Li B., Hou L., Xing P., Chen L., Wang C, & Xie S. (2023). NUAK1 promotes tumor metastasis through upregulating slug transcription in esophageal squamous cell carcinoma. Cancer Cell International, 23, 258.

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Western Blot Antibody Validation

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Foxo1, pFoxo1-S²⁵³, Akt, pAkt-T³⁰⁸, ERK1/2, pERK1/2-T²⁰²Y²⁰⁴, GAPDH, and α-actin antibodies were from Cell Signaling Technology (Billerica, MA), and Agt antibody was purchased from Immuno-Biological laboratories, Inc (Japan). Insulin and collagenase were purchased from Sigma, and Percoll from Amersham.

Qi Y., Zhang K., Wu Y., Xu Z., Yong Q., Kumar R., Baker K.M., Zhu Q., Chen S, & Guo S. (2014). Novel Mechanism of Blood Pressure Regulation By Foxo1-Mediated Transcriptional Control of Hepatic Angiotensinogen. Hypertension, 64(5), 1131-1140.

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Western Blot Analysis of Signaling Proteins

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Cultured cells were homogenized in RIPA buffer (Cell Signaling) containing protease inhibitors (Complete; Roche, IN, USA). Protein concentrations were measured using the Bradford protein assay (Bio-Rad), according to the manufacturer’s instructions. Protein samples (5 µg) was boiling for 5 min, then subjected to 13- or 17-wells of 5%–20% SDS-polyacrylamide Supersep Ace (Wako) gel electrophoresis. Gels that contains separated products were transferred to nitrocellulose membranes, followed by blocking with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20. Nitrocellulose membranes then incubate overnight with primary antibodies for p-FAK (Y397) (1:1000, Cell Signaling), FAK (1:1000, Cell Signaling), p-ERK1/2 (T202/Y204) (1:1000, Cell Signaling), ERK1/2 (1:1000, Cell Signaling), p-Akt (S473) (1:1000, Cell Signaling), Akt (1:1000, Cell Signaling), and GAPDH (1:1000, Proteintech, IL, USA). After washing with Tris-buffered saline containing 0.1% Tween 20, membranes were incubated with secondary antibody for anti-rabbit or anti-mouse IgG horseradish peroxidase-conjugated (1:3000, Cell Signaling) for one hour at room temperature. Bands of target proteins were detected using an ECL detection system (Wako). ImageJ Fiji (NIH) was used to analyses the bands. Data are showed as the mean ± standard error of the mean (SEM). GAPDH was used as the loading control.

Ariyani W., Miyazaki W., Amano I, & Koibuchi N. (2022). Involvement of integrin αvβ3 in thyroid hormone-induced dendritogenesis. Frontiers in Endocrinology, 13, 938596.

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Comprehensive Immunoblot Analysis

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Immunoblot analysis was performed as previously described (7 (link)). Primary Abs against p-ERK1/2 T202/Y204 (catalog 4370), total ERK1/2 (catalog 4695), p-AKT S473 (catalog 4060), total AKT (catalog 9272), p-Rb S807/811 (catalog 8516), total Rb (catalog 9309), p-p90RSK T359/S363 (catalog 9344), RSK1 (catalog 8408), p-HER2/ErbB2 Y1221/1222 (catalog 2243), HER2/ErbB2 (catalog 2165), p-EGFR Y1068 (catalog 3777), cyclin D1 (catalog 2922), CDK4 (catalog 12790), CDK6 (catalog 13331), vinculin (catalog 18799), and β-actin (catalog 12620) were purchased from Cell Signaling Technology. Pierce HRP-conjugated secondary Abs (anti-rabbit and anti-mouse) were purchased from Thermo Fisher Scientific. Amersham ECL Prime chemiluminescent detection reagent (GE Healthcare Life Sciences) was used to visualize protein expression.

Li T., Kikuchi O., Zhou J., Wang Y., Pokharel B., Bastl K., Gokhale P., Knott A., Zhang Y., Doench J.G., Ho Z.V., Catenacci D.V, & Bass A.J. (2023). Developing SHP2-based combination therapy for KRAS-amplified cancer. JCI Insight, 8(3), e152714.

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Western Blot Analysis of Signaling Proteins

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Western blot analyses were performed as described previously (25 (link)). Antibodies used in this study were as follows: MET (sc-161), ERK1 (sc-94), and PARP (sc-7150) from Santa Cruz (Santa Cruz, CA, USA); p-EGFR (Y1068) (#2237), EGFR (#4405), p-MET (Y1234/Y1235) (#3123), phospho-AKT (Ser473) (#9271), AKT (#9272), p-ERK1/2 (T202/Y204) (#4370), p-p70 S6K (T389) (#9205), p70 S6K (#9202), p-S6 (S235/S236) (#4856), S6 (#2217) and XIAP (#2045) from Cell Signaling (Danvers, MA, USA); α-tubulin, β-actin, and horseradish peroxidase-conjugated secondary antibodies from Sigma.

YI Y.W., YOU K., BAE E.J., KWAK S.J., SEONG Y.S, & BAE I. (2015). Dual inhibition of EGFR and MET induces synthetic lethality in triple-negative breast cancer cells through downregulation of ribosomal protein S6. International Journal of Oncology, 47(1), 122-132.

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Western Blot Analysis of Signaling Pathways in Purified Immune Cells

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Purified B cells or T-cells were lysed using 20 mM Tris-HCl (pH7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 μg/ml leupetin, 1% Triton X-100, 1 mM PMSF and 1 mM Na₃VO₄. Total protein was quantified by the Bradford assay, and 10 μg was loaded per lane onto SDS-PAGE gels. The following primary Abs were obtained from Cell Signaling Technology: p-STAT3^S727 (#9134), P-Erk1,2^T202/Y204 (#4376), Erk1,2 (#4695), p-NF-κB^S536 (#3031), p-MEK1/2^S217/221 (#9121), MEK1/2 (#9122), AKT (#9272), p-AKT^T308 (#9275), α-tubulin (#2144). β-actin (Advanced Immunochemical Inc, catalog # RGM2), Bcl-2 (Santa Cruz Biotechnology, Inc., # sc-23960). Antibodies to Nrf2 (ab71890), NQO1 (ab2346), GCLC (ab53179) and GCLM (ab81445) were purchased from Abcam. HRP-conjugated secondary antibodies and the ECL-plus detection kit (Amersham) were used to develop the blot. For western blot analysis of purified B and T-cells, cells were pooled from 3-4 mice for each lane. The respective band intensities were measured using ImageJ software version 1.37 (http://rsb.info.nih.gov/ij), and normalized against the corresponding β-actin or α-tubulin levels. Where samples from different strains were compared, normalized band intensities were expressed as ratios, relative to the corresponding B6 levels.

Wu T., Ye Y., Min S.Y., Zhu J., Khobahy E., Zhou J., Yan M., Hemachandran S., Pathak S., Zhou X.J., Andreeff M, & Mohan C. (2014). Targeting multiple signaling axes and oxidative stress using a synthetic triterpenoid prevents murine lupus nephritis. Arthritis & rheumatology (Hoboken, N.J.), 66(11), 3129-3139.

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3T3-L1 Adipocyte Differentiation Protocol

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3T3-L1 preadipocytes were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and penicillin-streptomycin were purchased from Mediatech (Herndon, VA, USA). Oil red O solution, isobutylethylxanthine (IBMX), dexamethasone, and insulin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies including phospho (P)-Akt1 (T308), total (T)-Akt1, P-ERK1/2 (T202/Y204), T-ERK1/2, C/EBPα, PPARγ, Fatty acid synthase (FAS), Acetyl-CoA carboxylase (ACC) and GAPDH were purchased from Cell Signaling Technology (Boston, MA, USA).

Chayaratanasin P., Caobi A., Suparpprom C., Saenset S., Pasukamonset P., Suanpairintr N., Barbieri M.A, & Adisakwattana S. (2019). Clitoria ternatea Flower Petal Extract Inhibits Adipogenesis and Lipid Accumulation in 3T3-L1 Preadipocytes by Downregulating Adipogenic Gene Expression. Molecules, 24(10), 1894.

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