Mouse anti phospho erk1 2

Manufactured by Cell Signaling Technology

Sourced in United States

The Mouse anti-phospho-ERK1/2 is a primary antibody that detects endogenous levels of ERK1 and ERK2 only when dually phosphorylated at Thr202/Tyr204 of ERK1 and Thr185/Tyr187 of ERK2. This antibody is specific for the activated, phosphorylated form of ERK1/2.

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Lab products found in correlation

Rabbit anti erk1 2, by Cell Signaling Technology (3 mentions) Rabbit anti phospho p38, by Cell Signaling Technology (2 mentions) Mouse anti gapdh antibody, by Abcam (1 mentions) Ripa lysis buffer, by Merck Group (1 mentions) Rabbit anti akt, by Cell Signaling Technology (1 mentions) Epidermal growth factor (egf), by Merck Group (1 mentions) Human sdf 1α, by Thermo Fisher Scientific (1 mentions) Human allophycocyanin conjugated cd44, by BD (1 mentions) Growth factor reduced matrigel, by BD (1 mentions) Mouse anti phospho akt, by Cell Signaling Technology (1 mentions) Gallein, by Tokyo Chemical Industry (1 mentions) Complete mini, by Roche (1 mentions) Supersignal west dura chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Polyvinylidene fluoride membrane, by Merck Group (1 mentions) Phosstop, by Roche (1 mentions) Mouse anti erk1 2, by Cell Signaling Technology (1 mentions) β mercaptoethanol, by Merck Group (1 mentions) Amersham hyperfilm ecl, by GE Healthcare (1 mentions) Donkey anti mouse secondary antibodies, by Jackson ImmunoResearch (1 mentions) Anti erk1 2, by Cell Signaling Technology (1 mentions)

Spelling variants of this product

ERK1/2 (1487 citations) Anti-ERK1/2 (692 citations) Phospho-ERK1/2 (582 citations) Anti-phospho-ERK1/2 (429 citations) Mouse anti-ERK1/2 (15 citations) Mouse monoclonal anti-phospho-ERK1/2 (3 citations) Mouse phospho-ERK1/2 (2 citations) Anti-phospho-ERK1/2 MAPK (T202/Y204) mouse antibody (2 citations) Mouse monoclonal anti Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (2 citations)

12 protocols using mouse anti phospho erk1 2

Western Blot Analysis of FGF-18 Signaling

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Radio immunoprecipitation assay (RIPA) lysis buffer (Sigma, St. Louis, MO, USA) was used to split cells. Protein samples were subjected to 10% dodecyl sulfate, sodium salt (SDS)-Polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane, then blocked for 1 h at room temperature. Each membrane was incubated with primary antibodies at 4 °C overnight and then secondary antibodies at room temperature for 1 h the next day. The following antibodies were used: mouse anti-FGF-18 antibody (Absci, Vancouver, WA, USA; dilution rates of 1:1000), mouse anti-FGFR3 antibody (Abcam, Cambridge, UK; dilution rates of 1:1000), mouse anti anti-phospho-FGFR3 antibody (Abcam; dilution rates of 1:1000), mouse anti-ERK1/2 antibody (Abcam, dilution rates of 1:1000), mouse anti-phospho-ERK1/2 (Cell Signaling Technologies, Danvers, MA, USA; dilution rates of 1:1000), and mouse anti-GAPDH antibody (Abcam, concentration of 0.5 µg/mL).

Wang Y., Yang T., Liu Y., Zhao W., Zhang Z., Lu M, & Zhang W. (2017). Decrease of miR-195 Promotes Chondrocytes Proliferation and Maintenance of Chondrogenic Phenotype via Targeting FGF-18 Pathway. International Journal of Molecular Sciences, 18(5), 975.

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Signaling Pathways in Cancer Stem Cells

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LPA, EGF, IGF and PTx were from Sigma. Human SDF1-α was from Pepro Tech. Growth-factor-reduced Matrigel was from BD Biosciences. Rabbit anti-AKT, mouse anti-phospho-AKT, rabbit anti-ERK1/2, and mouse anti-phospho-ERK1/2 antibodies were from Cell Signaling Technology. Mouse anti-mortalin was from NeuroMab. Mouse anti-Gαt was a gift from Dr. Heidi Hamm (Vanderbilt University). Human allophycocyanin-conjugated CD44 was from BD Biosciences, and human phycoerythrin-conjugated CD133 was from Miltenyl Biotech. Paclitaxel was from LC Laborateries. Gallein was from TCI America.

Paudyal P., Xie Q., Vaddi P.K., Henry M.D, & Chen S. (2017). Inhibiting G protein βγ signaling blocks prostate cancer progression and enhances the efficacy of paclitaxel. Oncotarget, 8(22), 36067-36081.

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Retinal Protein Extraction and Quantification

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Retinal explants were snap frozen in liquid nitrogen immediately after cultivation, and stored at −80 °C. Individual retina were lysed in 1 × LDS sample buffer (Thermo Scientific) containing 3% b-mercaptoethanol (Sigma), protease inhibitors (cOmplete mini, Roche), and phosphatase inhibitors (PhosSTOP, Roche) on ice for 20 minutes, sonicated, and clarified by centrifugation at 16,000 × g for 10 min at 4 °C. Lysates were boiled for 5 minutes and separated on 4–12% NuPage gels, transferred to polyvinylidene fluoride membranes (Millipore), and probed with either mouse anti-ERK1/2 (#4696, Cell Signaling) or mouse anti-phospho ERK1/2 (#5726, Cell Signaling) antibodies. Immunoreactive bands were detected using donkey anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories) and SuperSignal West Dura Chemiluminescent Substrate (Thermo Scientific), then captured on Amersham Hyperfilm ECL (GE Healthcare). Immunoreactive bands were quantified using Fiji software (https://fiji.sc) and the ratio of P-ERK1/2 to ERK1/2 was determined for individual retinas.

Sardar Pasha S.P., Münch R., Schäfer P., Oertel P., Sykes A.M., Zhu Y, & Karl M.O. (2017). Retinal cell death dependent reactive proliferative gliosis in the mouse retina. Scientific Reports, 7, 9517.

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Pathway Analysis of PTN Signaling

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Goat anti-PTN was obtained from Abcam. Mouse anti-phospho ERK1/2 and anti-ERK1/2 were obtained from Cell Signaling Technology. Mouse anti-PTPRZ1 was obtained from BD. HRP-conjugated anti-goat antibodies were obtained from Santa-Cruz Biotechnology. HRP-conjugated anti-mouse antibodies were obtained from Bio-Rad. Recombinant human PTN was obtained from R&D Systems. Mouse anti-β-actin antibody was obtained from Sigma.

Sethi G., Kwon Y., Burkhalter R.J., Pathak H.B., Madan R., McHugh S., Atay S., Murthy S., Tawfik O.W, & Godwin A.K. (2014). PTN Signaling: Components and Mechanistic Insights in Human Ovarian Cancer. Molecular carcinogenesis, 54(12), 1772-1785.

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Evaluating NRAS Mutant Signaling in Cells

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COS-7 (ATCC CRL-1651) and 293FT (ThermoFisher Scientific R7007) cells were cultured in DMEM medium (Quality Biologicals) containing 10% FBS. Where indicated, cells were treated with recombinant human EGF protein (R&D, Cat# 236-EG-200) in medium free of FBS. NRAS constructs (wild-type, p.G12V (c.G35T), and p.D57A (c.A170C) were transfected into COS7 cells using PolyFect Transfection reagent (Qiagen, Cat# 301105) while the transfection of 293FT cells was performed using Lipofectamine 2000 from Invitrogen. Two days after transfection, total cell lysates were generated in RIPA buffer (Thermo Scientific) and subjected to water bath sonication (Bioruptor) Samples were resolved in NuPage 4–12% Bis-Tris gel (Invitrogen) electrophoresis. The primary antibodies used were mouse anti-β-actin (Sigma, A5316), mouse anti-Pan AKT (cell signaling, 2920S), rabbit anti-Phospho AKT(S473) (cell signaling, 4060S), rabbit anti-total ERK1/2 (cell signaling, 9106S), mouse anti-Phospho ERK1/2 (Cell signaling, 4695S), and mouse anti-RAS (Millipore Cat# 05–516). To determine GTP-bound NRAS level in cells expressing WT or mutant NRAS proteins, RAS-GTP was pulled down by RAF1-RBD conjugated agarose beads followed by western by anti-RAS antibody (Millipore, Cat17-218).

Brown K.M., Xu M., Sargen M., Jang H., Zhang M., Zhang T., Zhu B., Jones K., Kim J., Mendoza L., Hayward N.K., Tucker M.A., Goldstein A.M., Yang X.R., Stewart D.R., Hicks B., Consonni D., Pesatori A.C., Fargnoli M.C., Peris K., Stratigos A., Menin C., Ghiorzo P., Puig S., Nagore E., Andresson T., Nussinov R., Calista D, & Landi M.T. (2021). Novel MAPK/AKT-impairing germline NRAS variant identified in a melanoma-prone family. Familial cancer, 21(3), 347-355.

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Immunoblotting of Phosphorylated ERK

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Primary antibodies used: 1:2000 dilution rabbit anti-NRMT1 [1 (link)], 1:5000 dilution mouse anti-tubulin (NeoMarkers, Fremont, CA), and 1:3000 dilution rabbit anti-GAPDH (Trevigen, Gaithersburg, MD). For phospho-ERK immunoblots, cells were starved for 24 hours in serum free media with 1% bovine serum albumin. The next day, the media was changed to that containing 5% FBS and cells were lysed after 15 minutes. Cell lysis buffer included the phosphatase inhibitors sodium fluoride (50 mM), β-glycerophosphate (10 mM), and sodium orthovanadate (0.2 mM, all Sigma). 1:2000 dilution mouse anti-phospho-ERK1/2 (Cell Signaling, Danvers, MA) and 1:1000 dilution rabbit anti-ERK (generous gift from Dr. Alan Cheng) were used as primary antibodies.

Bonsignore L.A., Butler J.S., Klinge C.M, & Tooley C.E. (2015). Loss of the N-terminal methyltransferase NRMT1 increases sensitivity to DNA damage and promotes mammary oncogenesis. Oncotarget, 6(14), 12248-12263.

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Immunoblotting of Intestinal Epithelial Proteins

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Polyacrylamide gels (10%) were loaded with 40μg of mouse epithelial intestinal protein. Immunoblotting was performed using rabbit anti-Erk1/2 (Cell Signaling Technology #4696), mouse anti-phospho-Erk1/2 (Cell Signaling Technology #9101), and mouse anti-α-Tubulin (Sigma-Aldrich #T6074). Membranes were incubated with primary antibodies overnight at 4°C and incubated for 1 hour at room temperature with secondary antibodies (Rockland) diluted at 1:10,000. Membranes were scanned using a LI-COR Odyssey infrared imaging system.

Gierut J.J., Lyons J., Shah M.S., Genetti C., Breault D.T, & Haigis K.M. (2015). Oncogenic K-Ras Promotes Proliferation in Quiescent Intestinal Stem Cells. Stem cell research, 15(1), 165-171.

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Immunohistochemical Profiling of Microglia and Astrocytes

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Antibodies used for immunohistochemical staining include rabbit anti-p47phox antibody
(sc-14015; Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-phospho-ERK1/2
(phosphorylated extracellular signal-regulated kinases 1/2) monoclonal antibody (9102;
Cell Signaling, Beverly, MA). Cell-type-specific antibodies include rat anti-CD11b
(cluster of differentiation molecule 11b, 550274; BD Biosciences, San Jose, CA) and rabbit
anti-Iba-1 (ionized calcium-binding adapter molecule-1) antibodies (019-19741; Wako
BioProducts, Richmond, VA) for microglia, and rabbit anti-glial fibrillary acidic protein
(GFAP) antibodies (G9269; Sigma-Aldrich, St. Louis, MO) for astrocytes. Secondary
antibodies include goat anti-mouse IgG-Alexa488 (A11001), goat anti-rabbit IgG Alexa 488
(A110034), goat anti-mouse IgG Alexa fluor 594 (A11005), and goat anti-rat IgG Alexa fluor
594 (A11007; Life Technologies/Invitrogen, Carlsbad, CA).

Chuang D.Y., Cui J., Simonyi A., Engel V.A., Chen S., Fritsche K.L., Thomas A.L., Applequist W.L., Folk W.R., Lubahn D.B., Sun A.Y., Sun G.Y, & Gu Z. (2014). Dietary Sutherlandia and Elderberry Mitigate Cerebral Ischemia-Induced Neuronal Damage and Attenuate p47phox and Phospho-ERK1/2 Expression in Microglial Cells. ASN NEURO, 6(6), 1759091414554946.

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Endothelial Cell VEGF Signaling Pathway

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Antibodies were goat anti-VEGFR2 (R&D Systems, Minneapolis, MN), rabbit anti-ERK1/2, mouse anti–phospho-ERK1/2 (Thr²⁰²/Tyr²⁰⁴), rabbit anti-p38, rabbit anti–phospho-p38 (Thr¹⁸⁰/Tyr¹⁸²), rabbit anti–phospho-VEGFR2 (Tyr¹¹⁷⁵), rabbit anti–ATF-2, rabbit anti–phospho-ATF-2 (Thr⁷¹), rabbit anti-NRP1 (Cell Signaling Technology, Danvers, MA), mouse anti–α-tubulin, mouse anti–PECAM-1 (CD31; Santa Cruz Biotechnology, Santa Cruz, CA), and mouse anti–VCAM-1 (DAKO, Glostrup, Denmark).
Reagents were as follows. Endothelial cell growth medium (ECGM) was from PromoCell (Heidelberg, Germany). Scrambled, ATF-2, NRP1, and VCAM1 siRNA duplexes were purchased as siGENOME SMARTpools from Dharmacon (Thermo Scientific, Lafayette, CO) unless otherwise stated in the figure legends. Recombinant human VEGF-A₁₆₅ was from Genentech (San Francisco, CA), and VEGF-A₁₂₁ was from Promocell.

Fearnley G.W., Odell A.F., Latham A.M., Mughal N.A., Bruns A.F., Burgoyne N.J., Homer-Vanniasinkam S., Zachary I.C., Hollstein M.C., Wheatcroft S.B, & Ponnambalam S. (2014). VEGF-A isoforms differentially regulate ATF-2–dependent VCAM-1 gene expression and endothelial–leukocyte interactions. Molecular Biology of the Cell, 25(16), 2509-2521.

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Western Blot Analysis of Phosphorylated Signaling Proteins

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Washed platelets were lysed in cold RIPA buffer (62.5 mM TRIS HCl, 100 mM NaCl, 1% NP-40, 0.1% 13 Tween 20, 1 mM Na-orthovanadate, 1 mM PMSF, 10 mM Na-pyrophosphate, 10 mM NaF 14 pH 8.0 and protease inhibitor cocktail), sonicated and protein yield was quantified using the BCA protein assay kit (Sigma-Aldrich, St. Louis, MO, USA). Twenty micrograms of protein samples were prepared with the Laemmli method, and equivalent amounts of protein were separated on 12% SDS-PAGE gels, transferred to nitrocellulose membrane and bands of interest detected using rabbit anti-phospho p38, mouse anti-phospho ERK1/2 and rabbit anti-phospho Akt (1:1000; Cell Signalling technology, Danvers, MA, USA). Then membranes were incubated with peroxidase-conjugated secondary antibody (goat anti-rabbit from BioRad laboratories, Milan, Italy and anti-mouse from Sigma-Aldrich, St. Louis, MO, USA). Immunoreactive bands were visualized by enhanced chemiluminescence (GE healthcare, Chicago, IL, USA) and analysed with the QuantityOne software (Bio-Rad Laboratories, Milan, Italy) for densitometric analysis including normalization for total protein loading visualized with MEMcode (Bio-Rad Laboratories, Milan, Italy) [28 (link)].

Banfi C., Amadio P., Zarà M., Brioschi M., Sandrini L, & Barbieri S.S. (2022). Prenylcysteine Oxidase 1 (PCYOX1), a New Player in Thrombosis. International Journal of Molecular Sciences, 23(5), 2831.

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