Phospho erk1 2t202 y204

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-ERK1/2T202/Y204 is a product for the detection of phosphorylated extracellular signal-regulated kinase 1 and 2 (ERK1/2) at threonine 202 and tyrosine 204 residues. It is a laboratory research tool used to measure the activation status of the ERK1/2 signaling pathway.

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Lab products found in correlation

44 protocols using phospho erk1 2t202 y204

Investigating EGFR and FGF Signaling Pathways

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The following antibodies were used: phospho EGFR Y1068 (Abcam AB5644),
phospho EGFR Y1068 (Cell Signaling 3777), EGFR (Cell Signaling 2646), EGFR (Cell
Signaling 4267), phospho ERK1/2 T202/Y204 (Cell Signaling 9101), phospho ERK1/2,
T202/Y204 (Cell Signaling 4370), ERK1/2 (Cell Signaling 9102), phospho AKT S473
(Cell Signaling 4060), AKT1/2/3 (Santa Cruz sc-8312), BIM (Cell Signaling 2933),
Actin (Cell Signaling 4970), Actin-HRP conjugated (Cell Signaling 12262), FGFR1
(Cell Signaling 9740), and FGFR3 (Cell Signaling 4574), phospho FRS2α
Y436 (Cell Signaling 3861), E-Cadherin (Cell Signaling 3195), N-Cadherin (Cell
Signaling 13116), Zeb1 (Cell Signaling 3396), Vimentin (Cell Signaling 5741).
Gefitinib, WZ4002, AZD9291, and BGJ398 (all from Selleck) were dissolved in DMSO
to a final concentration of 10 mmol/liter and stored at −20 °C.
The 18 drugs tested in the long-term assay are listed in Supplemental Table 3.

Raoof S., Mulford I.J., Frisco-Cabanos H., Nangia V., Timonina D., Labrot E., Hafeez N., Bilton S.J., Drier Y., Ji F., Greenberg M., Williams A., Kattermann K., Damon L., Sovath S., Rakiec D.P., Korn J.M., Ruddy D.A., Benes C.H., Hammerman P.S., Piotrowska Z., Sequist L.V., Niederst M.J., Barretina J., Engelman J.A, & Hata A.N. (2019). Targeting FGFR overcomes EMT-mediated resistance in EGFR mutant non-small cell lung cancer. Oncogene, 38(37), 6399-6413.

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Investigating EGFR and FGF Signaling Pathways

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Western Blot Analysis of Signaling Proteins

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Western blots were performed following the method from another literature published by Novartis^{58 (link)}: total cell lysates were prepared by direct lysis of cells with RIPA buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitors (Thermo Fisher Scientific) with incubation on ice for 20 min and followed by centrifugation at 12,000×g at 4 °C for 5 min. The supernatant was collected and protein concentration was determined using Pierce BCA protein assay kit (#23225, Thermo Fisher Scientific). Equal amounts of proteins were resolved by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes and probed by the following antibodies with indicated dilutions: phospho-ERK1/2 (T202/Y204) (9101, Cell Signaling Technology, 1:1000), total ERK1/2 (9102, Cell Signaling Technology, 1:1000), SHP2 (3397, Cell Signaling Technology, 1:1000), phospho-STAT1 (Y701) (9167, Cell Signaling Technology, 1:1000), total STAT1 (14994, Cell Signaling Technology, 1:1000), Vinculin (13901, Cell Signaling Technology, 1:1000). After detection of phospho-STAT1 and phospho-ERK1/2, the PVDF membranes were incubated with Restore PLUS Western Blot Stripping Buffer (#46430, Thermo Fisher Scientific) before probing for levels of total STAT1 and total ERK1/2.

Wang Y., Mohseni M., Grauel A., Diez J.E., Guan W., Liang S., Choi J.E., Pu M., Chen D., Laszewski T., Schwartz S., Gu J., Mansur L., Burks T., Brodeur L., Velazquez R., Kovats S., Pant B., Buruzula G., Deng E., Chen J.T., Sari-Sarraf F., Dornelas C., Varadarajan M., Yu H., Liu C., Lim J., Hao H.X., Jiang X., Malamas A., LaMarche M.J., Geyer F.C., McLaughlin M., Costa C., Wagner J., Ruddy D., Jayaraman P., Kirkpatrick N.D., Zhang P., Iartchouk O., Aardalen K., Cremasco V., Dranoff G., Engelman J.A., Silver S., Wang H., Hastings W.D, & Goldoni S. (2021). SHP2 blockade enhances anti-tumor immunity via tumor cell intrinsic and extrinsic mechanisms. Scientific Reports, 11, 1399.

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Western Blot Analysis of Signaling Pathways

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For each sample, 20 μg of protein was separated by SDS/PAGE and transferred to PVDF membranes.30 The membranes were blocked for 1 hour at room temperature with 5% powdered skim milk in Tris‐buffered saline pH 7.4 with 0.05% Tween (TBST) and incubated with antibodies for TGFBR2 (#SC400; 1:1000; Santa Cruz Biotechnology, Dallas, TX), phospho‐SMAD2 (S465‐467, #3101S; 1:1000; Cell Signaling, Danvers, MA), SMAD2 (#3122S; 1:1000; Cell Signaling), phospho‐ERK1/2 (T202‐Y204, #9101S; 1:1000; Cell Signaling), ERK1/2 (#9102S; 1:1000; Cell Signaling), phospho‐p38 (T180‐Y182, #4511S; 1:2000; Cell Signaling), p38 (#9212S; 1:2000; Cell Signaling), or β‐actin (#A5316; 1:5000; Sigma, St. Louis, MO). Antibodies were diluted in TBST with 5% milk and incubated overnight at 4°C. Blots were then washed 3 times (5 minutes each) in TBST and incubated with HRP‐conjugated secondary antibody (goat antirabbit or antimouse; Biorad, Hercules, CA; #170‐6515 or #170‐6516; 1:3000‐1:5000 in TBST with 5% milk) for 1 hour at room temperature. Bound antibodies were detected using enhanced chemiluminescence (ECL) (Thermo, Rockford, IL). Band density was analyzed by densitometry, using the Image J software Version 1.48 (NIH, Bethesda, MD).

Wei H., Hu J.H., Angelov S.N., Fox K., Yan J., Enstrom R., Smith A, & Dichek D.A. (2017). Aortopathy in a Mouse Model of Marfan Syndrome Is Not Mediated by Altered Transforming Growth Factor β Signaling. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 6(1), e004968.

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Protein Extraction and Analysis Protocol

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MV4;11 and 293T cells were lysed with RIPA buffer supplemented with halt protease inhibitor cocktail (Thermo Fisher Scientific) and 0.1% benzonase (Novagen) on ice for 60 minutes. Lysates were clarified by centrifugation at 20,000 xg for 10 minutes at 4 ºC. NIH/3T3 and 293FT cells were lysed with RIPA buffer supplemented with cOmplete protease inhibitors (Roche) and PhosSTOP phosphatase inhibitors (Roche) on ice for 60 minutes. Lysates were clarified by centrifugation at 20,000 xg for 10 minutes at 4 ºC. Protein concentration was determined by a BCA assay (Pierce), and all samples were run with equal total protein content. The following antibodies were used in this study: HA (Cell Signaling, #3724 and #2367), BRD4 long isoform (Bethyl labs, #A301-985A), BRD4 short isoform (Abcam, #ab128874), BRD3 (Abcam, #ab56342), BRD2 (Bethyl labs, #A302–582A), FKBP12 (Abcam, #ab24373), vinculin (Santa Cruz, #Sc-25336), GAPDH (Millipore, #MAB374), phospho-AKT S473 (Cell Signaling, #4060), AKT (Cell Signaling, #2920), phospho-MEK1/2 S221 (Cell Signaling, #2338), MEK1/2 (Cell Signaling, #4694), phospho-ERK1/2 T202/Y204 (Cell Signaling, #4370), ERK1/2 (Cell Signaling, #4696), phospho-Pol II S2 (Millipore, #04-1571-1), and Pol II (Santa Cruz, #Sc-899). Imaging was performed by detection of fluorescently labelled infrared secondary antibodies (IRDye) on the Odyssey CLx Imager (LI-COR).

Nabet B., Roberts J.M., Buckley D.L., Paulk J., Dastjerdi S., Yang A., Leggett A.L., Erb M.A., Lawlor M.A., Souza A., Scott T.G., Vittori S., Perry J.A., Qi J., Winter G.E., Wong K.K., Gray N.S, & Bradner J.E. (2018). The dTAG system for immediate and target-specific protein degradation. Nature chemical biology, 14(5), 431-441.

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Protein Quantification and Western Blot Analysis

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The cells or tissues from in vitro or in vivo experiments were lysed in RIPA Buffer (1 mM EDTA pH 8.0, 50 mM Tris-HCl pH 8.0, 2% SDS, 5 mM DTT), and their protein concentration was determined by the BCA assay (Beyotime, Shanghai, China). The total protein (100 μg) was separated by 10%-15% SDS-PAGE gel and transferred to PVDF (polyvinylidene fluoride) membranes (Invitrogen, California, USA), and blocked with 5% skimmed milk powder in PBST (Phosphate Buffered Saline with Tween) or 5% w/v BSA, 1×TBS, 0.1% Tween at 4°C with gentle shaking about 12 hours. The membranes were immunoblotted with the primary antibodies for 4 hours at room temperature or overnight at 4°C and then incubated with the secondary antibodies for 2 hours. The corresponding semi-quantitative analysis was based on optical density with Image J software. In western blot analysis, cyclin B1, cyclin D1, BCL-2 (Boster Biological Technology), cyclin A, BAX, PARP, caspase-3, Vimentin (Proteintech), GAPDH (Bioworld), RRM2, E-cadherin, N-cadherin, phospho-AKT (T308), and phospho-ERK1/2 (T202/Y204) (Cell Signaling) were used for immunoblotting with secondary antibodies conjugated with horseradish peroxidase (Bioworld) and visualized with Bio-Rad ChemiDoc™ Imagers. All other chemical reagents were bought from Sigma (Shanghai, China).

Sun H., Yang B., Zhang H., Song J., Zhang Y., Xing J., Yang Z., Wei C., Xu T., Yu Z., Xu Z., Hou M., Ji M, & Zhang Y. (2019). RRM2 is a potential prognostic biomarker with functional significance in glioma. International Journal of Biological Sciences, 15(3), 533-543.

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Signaling Pathway Inhibitors in Cancer

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The following antibodies were used: phospho-ERK 1/2 (T202/Y204) (Cell Signaling Technology, Cat # 4377); total ERK1/2 (Santa Cruz, Dallas, TX, Cat # sc-94); Ki67 (Santa Cruz sc-7846); cyclin D1, p27, and β-actin from Cell Signaling. The BRAF V600E inhibitor vemurafenib and the MEK1/2 inhibitor AZD6244 were from Selleck Chemicals (Shanghai, China); vemurafenib and AZD6244 were dissolved in dimethylsulfoxide (DMSO) (Fisher Scientific, Hangzhou, China) to generate a 10-mm stock. DMSO alone was used as the vehicle control at a concentration of 0.0005% (unless otherwise noted, all percentages represent volume-to-volume ratio), which is the highest concentration of DMSO in studies using vemurafenib and AZD6244.

Song H., Zhang J., Ning L., Zhang H., Chen D., Jiao X, & Zhang K. (2018). The MEK1/2 Inhibitor AZD6244 Sensitizes BRAF-Mutant Thyroid Cancer to Vemurafenib. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 24, 3002-3010.

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Cell Treatment and Protein Analysis

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Cells were treated with XMU‐MP‐5 and crizotinib at indicated doses for 4 h, then washed on ice with cold PBS before the addition of lysis buffer (3 M NaCl, 1 M Tris‐HCl, pH 7.5, 1% Triton X‐100, 5% glycerol) supplemented with protease inhibitor 50 mM NaF, 1 mM Na₃VO₄, 5 μg/ml Leupeptin, and 1mM PMSF. After scraping, cell lysates were sonicated then centrifuged 12,000 g for 30 min at 4°C. The protein concentrations were measured by BCA protein assay kit (Cat# P0012S, Beyotime, Shanghai, China). Antibodies of phospho‐ALK (Y1604) (Cat# 3341,1:1,000 dilution), ALK (Cat# 3633P, 1:1,000 dilution), phospho‐STAT3 (Y705) (Cat #9131, 1:1,000 dilution), STAT3 (Cat# 9139, 1:1,000 dilution), phospho‐AKT (S473) (Cat# 4060S, 1:1,000 dilution), AKT (Cat# 4691S, 1:1,000 dilution), phospho‐ERK1/2 (T202/Y204) (Cat# 9101L, 1:1,000 dilution), and ERK1/2 (Cat# 9102, 1:1,000 dilution) were purchased from Cell Signaling Technology. β‐actin (Cat# A5316, 1:5,000 dilution) was purchased from Sigma Aldrich.

Lu Y., Fan Z., Zhu S., Huang X., Zhuang Z., Li Y., Deng Z., Gao L., Hong X., Zhang T., Li L., Sun X., Huang W., Zhang J., Liu Y., Zhang B., Jiang J., Gui F., Wang Z., Li Q., Song S., Huang X., Wu Q., Chen L., Zhou D., Zhang J., Yun C., Chen L, & Deng X. (2021). A new ALK inhibitor overcomes resistance to first‐ and second‐generation inhibitors in NSCLC. EMBO Molecular Medicine, 14(1), e14296.

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Western Blot Analysis of Cancer Signaling

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Western blot and protein detection was performed as previously described [43 (link)]. The following primary antibodies were used: RB, phospho-RB^S807/S811, CDC25C, phospho-AKT^S473, phospho-cyclin E1^T62, phospho-ERK1/2^T202/Y204, ERBB2, phospho-ERBB2^Y1221/1222 and MYC (Cell Signaling, Danvers, MA); BRCA1, p53, cyclin E, CDK1, CDK2, ETV4, ETV5 and HRAS (Santa Cruz Biotechnology, Santa Cruz, CA); Actin (Abcam, Cambridge, MA); GAPDH (Fitzgerald, Acton, MA); β-Actin (Sigma-Aldrich); PAX8 (Epitomics, Burlingame, CA);

Taylor-Harding B., Aspuria P.J., Agadjanian H., Cheon D.J., Mizuno T., Greenberg D., Allen J.R., Spurka L., Funari V., Spiteri E., Wang Q., Orsulic S., Walsh C., Karlan B.Y, & Wiedemeyer W.R. (2014). Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS. Oncotarget, 6(2), 696-714.

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EGFR Signaling Pathway Activation

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The cells were lysed in RIPA buffer supplement with leupeptin (0.5 μg/mL), aprotin (0.5 μg/mL), PMSF (1 mM), sodium orthovanadate (0.2 mM), and BGP (0.2 mM). Western blotting was then performed. Antibodies against TNS4 and V5-tag were purchased from Invitrogen. phospho-EGFR (Y1092), ERK, phospho-ERK1/2 (T202/Y204), and β-actin were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). Anti-EGFR antibodies were purchased from Bethyl (Montgomery, AL, USA).

Kim S., Kim N., Kang K., Kim W., Won J, & Cho J. (2019). Whole Transcriptome Analysis Identifies TNS4 as a Key Effector of Cetuximab and a Regulator of the Oncogenic Activity of KRAS Mutant Colorectal Cancer Cell Lines. Cells, 8(8), 878.

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