Pt202 y204 erk

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The PT202/Y204-ERK is a reagent that specifically detects phosphorylation of ERK1/2 at Thr202/Tyr204. It is used in immunoblotting applications to measure activation of the ERK signaling pathway.

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Lab products found in correlation

Ps473 akt, by Cell Signaling Technology (4 mentions) Pt183 y185 jnk, by Cell Signaling Technology (3 mentions) Ps32 36 iκbα, by Cell Signaling Technology (2 mentions) Ps176 180 ikkα β, by Cell Signaling Technology (2 mentions) Py416 c src, by Cell Signaling Technology (2 mentions) Pt180 y182 p38, by Promega (2 mentions) Mouse trip6, by BD (2 mentions) Traf6, by Santa Cruz Biotechnology (2 mentions) C src, by Santa Cruz Biotechnology (2 mentions) Histone h3, by Cell Signaling Technology (2 mentions) Flag tag (flag), by Merck Group (2 mentions) Gapdh, by Santa Cruz Biotechnology (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Vinblastine, by Selleck Chemicals (1 mentions) Jnk in 8, by Selleck Chemicals (1 mentions) Pt180 y182 p38, by Cell Signaling Technology (1 mentions) Antibodies to perk, by Merck Group (1 mentions) Rigosertib, by Selleck Chemicals (1 mentions) Anti mouse hrp na931v, by GE Healthcare (1 mentions) Anti rabbit hrp na934v, by GE Healthcare (1 mentions)

8 protocols using pt202 y204 erk

Immunoblotting, Co-IP, and Recombinant Protein Purification

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Experimental procedures of immunoblotting, co-immunoprecipitation, and purification of recombinant glutathione-S-transferase (GST)-tagged or non-tagged proteins were performed as described previously [19 (link), 34 (link)]. Immunoblotting was performed using antibody specific to human TRIP6, A20 (Bethyl Laboratories), pT183/Y185-JNK, pT202/Y204-ERK, pS32/36-IκBα, pS176/180-IKKα/β, pY416-c-Src, CYLD, IκBα, IKKβ, Histone H3 (Cell Signaling, Danvers, MA, USA), pT180/Y182-p38 (Promega, Madison, WI, USA), ERK, JNK, mouse TRIP6 (BD Biosciences, San Jose, CA, USA), HA (Cayman, Ann Arbor, MI, USA), FLAG (Sigma-Aldrich), GFP, TRAF6, p38, c-Src, GAPDH, GST, ubiquitin, or phosphotyrosine (Santa Cruz Biotechnology, Dallas, TX, USA).

Lin F.T., Lin V.Y., Lin V.T, & Lin W.C. (2016). TRIP6 antagonizes the recruitment of A20 and CYLD to TRAF6 to promote the LPA2 receptor-mediated TRAF6 activation. Cell Discovery, 2, 15048-.

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Ras/Raf/MAPK Signaling Pathway Antibodies

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Antibodies to B-Raf (H-145), C-Raf (C-12), β-Tubulin (H-235), and Sos1 (C-23) were from Santa Cruz Biotechnology; antibodies to pS217/221-MEK, pT202/Y204-ERK, pS473-AKT, pS/TP, pS257/T261-MKK4, pT183/Y185-JNK, pT180/Y182-p38, and pS621-C-Raf sites were from Cell Signaling Technologies; antibodies to pERK were from Sigma; antibodies to Ras were from BD Biosciences. The pS642P-C-Raf antibody has been previously described (Dougherty et al., 2005 (link)). Rigosertib, Paxlitaxel/Taxol, SB-590885 (RAFi), Vincristine, Vinblastine, SP-600125 (JNKi) and JNK-IN-8 were from SelleckChem; KG5 was from Tocris Bioscience; N-acetylcystine (NAC) was from Sigma-Aldrich. GST-RafRBD was from Millipore; GST-H-Ras^G12V was from Cell Sciences.

Ritt D.A., Blanco M.A., Bindu L., Durrant D.E., Zhou M., Specht S.I., Stephen A.G., Holderfield M, & Morrison D.K. (2016). Inhibition of Ras/Raf/MEK/ERK Pathway Signaling by a Stress-induced Phospho-regulatory Circuit. Molecular cell, 64(5), 875-887.

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Immunoblotting and Protein Purification Protocols

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Experimental procedures of immunoblotting, co-immunoprecipitation, and purification of recombinant glutathione-S-transferase (GST)-tagged or non-tagged proteins were performed as described previously [19 (link), 34 (link)]. Immunoblotting was performed using antibody specific to human TRIP6, A20 (Bethyl Laboratories), pT183/Y185-JNK, pT202/Y204-ERK, pS32/36-IκBα, pS176/180-IKKα/β, pY416-c-Src, CYLD, IκBα, IKKβ, Histone H3 (Cell Signaling, Danvers, MA, USA), pT180/Y182-p38 (Promega, Madison, WI, USA), ERK, JNK, mouse TRIP6 (BD Biosciences, San Jose, CA, USA), HA (Cayman, Ann Arbor, MI, USA), FLAG (Sigma-Aldrich), GFP, TRAF6, p38, c-Src, GAPDH, GST, ubiquitin, or phospho-tyrosine (Santa Cruz Biotechnology, Dallas, TX, USA).

Lin F.T., Lin V.Y., Lin V.T, & Lin W.C. (2016). TRIP6 antagonizes the recruitment of A20 and CYLD to TRAF6 to promote the LPA2 receptor-mediated TRAF6 activation. Cell Discovery, 2, 15048-.

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Western Blot Analysis of Cellular Signaling

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Whole-cell lysates were prepared with RIPA buffer supplemented with protease inhibitors (Roche), NaF (10mM), PMSF (1mM) and Na₃VO₄ (1mM) or cells were directly lysed in SDS sample buffer. Proteins were separated using SDS-PAGE and transferred to PVDF membranes. Antibodies used for immunoblot from Cell Signaling Technology were pS473 AKT (4051), AKT (4691), pT202/Y204 ERK (9101), ERK (9102), Trib2 (13533), and C/EBPα (8178). The antibody against TAL1 was purchased from Santa Cruz Biotechnology (sc-12984). β-actin (A5316; Sigma) was used as a loading control. Secondary anti-mouse-HRP (NA931V) and anti-rabbit-HRP (NA934V) were obtained from GE Healthcare. Blots were visualized with SuperSignal west pico chemilumenscence (34080; Thermo Scientific) or SuperSignal west femto chemilumenscence substrate (34095; Thermo Scientific).

Stein S.J., Mack E.A., Rome K.S., Pajcini K.V., Ohtani T., Xu L., Li Y., Meijerink J.P., Faryabi R.B, & Pear W.S. (2016). Trib2 Suppresses Tumor Initiation in Notch-Driven T-ALL. PLoS ONE, 11(5), e0155408.

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Rosiglitazone Signaling Pathway Analysis

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METH (purity > 99%) was purchased from the National Institutes for Food and Drug Control (Beijing, China). Protease inhibitor cocktail and radio immunoprecipitation assay (RIPA) lysis buffer were obtained from Sigma (St. Louis, MO, USA). The BCA Protein Assay Kit were purchased from Thermo Fisher (Rockford, IL, USA). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from HyClone Laboratories Inc. (Legan, UT, USA). The primary antibodies pS21-GSK3α, GSK3α, IRS1, pS473-AKT, AKT, pS9-GSK3β, GSK3β, pT202/ Y204-ERK and ERK were purchased from Cell Signaling Technology (1:1000, Danvers, MA, USA). The primary antibodies APP (Abcam, Cambridge, Cambs, UK), pY896-IRS1 (1:1000, Absci, Nanjing, China), pY1355-IR (1:500, Bioworld, China), pT205-tau (1:1000, Abcam), anti-GAPDH (1:10,000, Sigma-Aldrich, USA) were also used in the present work. Rosiglitazone (Sigma, Purity > 98%, CAS:122320-73-4) was dissolved in dimethyl sulfoxide as a stock solution and then diluted in DMEM to a final concentration of 50 μM.

Chen L., Zhou L., Yu P., Fang F., Jiang L., Fei J., Xiao H, & Wang J. (2019). Methamphetamine exposure upregulates the amyloid precursor protein and hyperphosphorylated tau expression: The roles of insulin signaling in SH-SY5Y cell line. The Journal of toxicological sciences, 44(7).

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Western Blot Antibody Detection Protocol

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FLAG-tagged PTP1B was detected with anti-FLAG antibody (20543-1-AP [Proteintech]). PTP1B was detected with anti-PTP1B clone FG6-1G (MABS197; Merck Millipore) or with clone H-135 (sc-14021) from Santa Cruz Biotechnology to detect specifically the C-terminal part. Anti-CD22 antibody was purchased from Novus Biological (clone 2H1C4), anti-CD79A (Igα; clone HM47) and anti-SYK (4D10.2) from BioLegend. The antibodies directed against; BTK-pY223, #5082; PLCγ1, #5690; PLCγ2, #3872; PLCγ2-pY759, #3874; SYK-p525/526, #2710; ERK, #4695; ERK-pT202/Y204, #4370; GRB2, #3972; and GAPDH, #2118 were all from Cell Signaling. Anti-phospho-CD22-pY807 (ab32040) antibody was purchased from Abcam. Anti-Myc-tag (66004-1-Ig), anti-APLP2 (15041-1-AP) and anti-CBL (25818-1-AP) were from Proteintech. Anti-BTK (sc-1107) and anti-BCAP (AF4857) antibodies were obtained from Santa Cruz Biotechnology and R&D Systems Inc., respectively. All antibodies were used according to the manufacturer’s instructions. For imaging, a ChemoCam (Intas) equipped with a full-frame 3.2 megapixel Kodak KAF-3200ME camera was used. Western blot signals were quantified with Quantity One 4.6.9 (Bio-Rad). For statistical analysis, two-sample t tests were performed over three replicates using Microsoft Excel 2013. Error bars represent the SEM.

Schwarz J.J., Grundmann L., Kokot T., Kläsener K., Fotteler S., Medgyesi D., Köhn M., Reth M, & Warscheid B. (2021). Quantitative proteomics identifies PTP1B as modulator of B cell antigen receptor signaling. Life Science Alliance, 4(11), e202101084.

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Molecular Profiling of Receptor Tyrosine Kinases

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Entrectinib, crizotinib, capmatinib, and afatinib were purchased from Selleck Chemicals. Antibodies to total AKT, AKT pS473, total ERK, ERK pT202/Y204, total ROS1, ROS1 pY2274, total MET, MET pY1234/1235, total AXL, total EGFR, EGFR pY1068, EGFR pY1086, EGFR pY1173, total HER2, HER2 pY1196, HER2 pY1221/1222, HER3 pY1289 were purchased from Cell Signaling Technologies. Antibody to GAPDH was purchased from Santa Cruz Biotechnologies Inc. MET and CEP7 interphase FISH probes were purchased from Abbott Molecular Inc. MET, CEP7, MYC, and CEP8 metaphase FISH probes were purchased from Empire Genomics.

Tyler L.C., Le A.T., Chen N., Nijmeh H., Bao L., Wilson T.R., Chen D., Simmons B., Turner K.M., Perusse D., Kasibhatla S., Christiansen J., Dudek A.Z, & Doebele R.C. (2022). MET gene amplification is a mechanism of resistance to entrectinib in ROS1+ NSCLC. Thoracic Cancer, 13(21), 3032-3041.

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Detailed Cell Line Characterization and Treatments

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H2228, H3122, and HCC78 were a kind gift from Dr. John D. Minna (The University of Texas, Southwestern Medical Center, Dallas, TX) and STE-1 were a kind gift from Dr. Christine M. Lovly (Vanderbilt University School of Medicine, Nashville, TN). ROS1 fusion cell lines CUTO2 (SDC4-ROS1), CUTO23 and CUTO27 (CD74-ROS1), and CUTO28 (TPM3-ROS1) were generated at the University of Colorado from patient tumor samples following informed consent under an IRB-approved protocol. ALK and ROS1 fusion cell lines were grown in RPMI supplemented with 10% FBS. H1299, A549, H460, H1650, HCC827 were obtained from the University of Colorado Cancer Center Tissue Culture Core and cultured in RPMI supplemented with 5% FBS. NIH3T3 cells were obtained from American Type Culture collection and grown in Dulbecco’s Modified Eagle’s Media with 5% FBS. Crizotinib (PF-02341066) was obtained from Pfizer, Inc. NVP-TAE684 and MG132 were purchased from Selleck Chemicals. Antibodies used were ALK pY1278/1282/1283 (#3983), total ALK (#3791), ERK pT202/Y204 (#9191), total ERK (#9107), c-MYC (#5605), c-MYC pS62 (#13748) were purchased from Cell Signaling Technology. MYCBP (ab172444) c-MYC pT58 (28842) was purchased from Abcam, and alpha-tubulin (sc-8035), was purchased from Santa Cruz biotechnology.

Pilling A.B., Kim J., Estrada-Bernal A., Zhou Q., Le A.T., Singleton K.R., Heasley L.E., Tan A.C., DeGregori J, & Doebele R.C. (2018). ALK is a critical regulator of the MYC-signaling axis in ALK positive lung cancer. Oncotarget, 9(10), 8823-8835.

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