Phospho erk

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom

Phospho-ERK is a laboratory product that detects and quantifies the phosphorylated form of Extracellular Signal-Regulated Kinase (ERK), a key signaling protein involved in cellular processes such as proliferation, differentiation, and survival. This product provides researchers with a tool to investigate the activation and regulation of the ERK signaling pathway.

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Lab products found in correlation

527 protocols using phospho erk

Evaluating Intestinal Polyp Formation in APC-Mutant Mice

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Animal procedures were performed in accordance with guidelines from the local animal ethics committee. Two cohorts with 15 C57BL/6J-APCmin/+ mice (Jackson Laboratory, Maine) each were fed control chow or PLX4720 (the laboratory tool compound for vemurafenib)-infused (high dose) chow (13 (link)) for 28 days. Upon sacrifice, small intestine and colon were cut open length-wise, and polyp number and size were evaluated in a blinded fashion for proximal small intestine (PSI), distal small intestine (DSI), and colon using a Nikon SMZ645 microscope. Wild type C57Bl6 mice were treated with control and PLX chow for 6 months and sacrificed. All gastrointestinal tracts were formalin fixed, and paraffin embedded. Immunohistochemistry for phospho-ERK, and beta-catenin, was performed as previously described (14 (link)) using the following antibodies: phospho-ERK (Cell Signaling 4370); beta-catenin (Cell Signaling 9562). Stained slides were evaluated for histological features of mouse intestinal polyps, the number of polyps, and nuclear phospho-ERK and β-catenin were scored in a blinded fashion. For the latter, a polyp with a well oriented section, permitting evaluation of luminal and basal epithelium and with the highest proportion of nuclear staining was given an estimated percentage of positive nuclei in the polyp.

Amaravadi R.K., Hamilton K.E., Ma X., Piao S., Del Portillo A., Nathanson K.L., Carlino M.S., Long G.V., Puzanov I., Xu X., Morrissette J.J., Tsai K.Y., Flaherty K.T., Sosman J.A., Goodman G.R., McArthur G.A., Rustgi A.K., Metz D.C., Schuchter L.M., Chapman P.B, & Sepulveda A.R. (2015). Multiple gastrointestinal polyps in patients treated with BRAF inhibitors. Clinical cancer research : an official journal of the American Association for Cancer Research, 21(23), 5215-5221.

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Western Blot Analysis of Inflammatory Signaling

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RAW264.7 cells and colon tissues were homogenized, and protein levels were quantified using BCA reagent (Beyotime, Shanghai, China). Nuclear and cytoplasmic extractions were performed as instructed by the manufacturer (Beyotime, Shanghai, China). Samples were loaded and electrophoresed in 10–12% SDS-PAGE and then transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat milk diluted in TBS-T for 2 h followed by incubation with primary antibodies. The following primary antibodies were used in this experiment: iNOS, COX-2, phospho-IKKα/β, IKKα/β, phospho-IκBα, IκBα, phospho-ERK ½, ERK ½, phospho-p38, p38, p65, phospho-c-Jun, c-Jun, c-Fos, β-tubulin, Lamin A/C, phospho-JNK, JNK, IRAK4, and β-actin at 1:1,000 dilution (Cell Signaling Technology, Danvers, MA, USA). Secondary antibodies included horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG at 1:2,500 dilution.

Lertnimitphun P., Jiang Y., Kim N., Fu W., Zheng C., Tan H., Zhou H., Zhang X., Pei W., Lu Y, & Xu H. (2019). Safranal Alleviates Dextran Sulfate Sodium-Induced Colitis and Suppresses Macrophage-Mediated Inflammation. Frontiers in Pharmacology, 10, 1281.

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Western Blot Analysis of Phospho-proteins

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Immunoblotting was performed as described previously.^{31 (link)} In brief, whole-cell lysates were prepared using boiling lysis buffer (1% SDS, 10 mM Tris·HCl, pH 7.4). Equal amounts of proteins were separated using Criterion or mini gradient polyacrylamide gels (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes. The following rabbit antibodies for: phospho-S6 (S235/236), phospho-S6 (S240/244), phospho ERK½, phospho-Thr 389 p70S6K, phospho-AKT (S473) and phospho-AKT (T308), phospho-IRS1 (S1101), phospho-IRS1(S636/639), IRS1 and mouse anti-S6 antibody were from Cell Signaling Biotechnology (Danvers, MA, USA). Rabbit anti-actin and mouse monoclonal anti-GAPDH antibodies were from Sigma-Aldrich and Invitrogen (Camarillo, CA, USA), respectively. Secondary anti-rabbit and anti-mouse HRP-conjugated antibodies were from Cell Signaling Biotechnology.

Leontieva O.V., Demidenko Z.N, & Blagosklonny M.V. (2014). Rapamycin reverses insulin resistance (IR) in high-glucose medium without causing IR in normoglycemic medium. Cell Death & Disease, 5(5), e1214-.

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Quantitative Western Blot Analysis

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BMMCs were homogenized, and protein levels were quantified using BCA reagent (Beyotime, Shanghai, China). Nuclear and cytoplasmic extractions were performed as instructed by the manufacturer (Beyotime, Shanghai, China). Samples were loaded and electrophoresed by 10% to 12% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked in 5% nonfat milk and diluted in TBS-T for 2 h, followed by incubation with primary antibodies. The following primary antibodies were used in this experiment, iNOS, COX-2, phospho-IKKα/β, IKKα/β, phospho-IκBα, IκBα, phospho-ERK½, ERK½, phospho-p38, p38, p65, phospho-c-Jun, c-Jun, c-fos, β-tubulin, lamin A/C, phospho-JNK, JNK, phospho-cPLA₂, 5-LO, and β-actin, at a 1:1000 dilution (Cell Signaling Technology, Danvers, MA, USA). Secondary antibodies included horseradish peroxide-conjugated goat anti-rabbit antibody and anti-mouse IgG at a 1:2500 dilution.

Lertnimitphun P., Zhang W., Fu W., Yang B., Zheng C., Yuan M., Zhou H., Zhang X., Pei W., Lu Y, & Xu H. (2021). Safranal Alleviated OVA-Induced Asthma Model and Inhibits Mast Cell Activation. Frontiers in Immunology, 12, 585595.

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Neurochemical Profile of Neuroinflammation

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Phospho-Erk (p44/42 mitogen activated protein kinase), Total Erk 1/2, and phospho-CEBP-β from Cell Signaling (Danvers, MA), Peripherin (Millipore, MA), choline acetyl transferase (Millipore, MA), SERT (Abcam), Hand-2 (Invitrogen), N-acetyl cysteine and fMLF (Sigma), ROSstar™ 550 (Hydrocyanine-3 probe) from LI-COR ^{20 (link), 21 (link)}.

Chandrasekharan B., Saeedi B.J., Alam A., Houser M., Srinivasan S., Tansey M., Jones R., Nusrat A, & Neish A.S. (2019). Interactions Between Commensal Bacteria and Enteric Neurons, via FPR1 Induction of ROS, Increase Gastrointestinal Motility in Mice. Gastroenterology, 157(1), 179-192.e2.

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Cell Proliferation and Apoptosis Assay

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A cell counting kit (CCK) for WST assay was purchased from Donginbiotech Co. (Seoul, Korea). Crystal violet solution was purchased from Sigma-Aldrich (St Louis, MO, USA). Anti-phospho-AMPK, phospho-p38, phospho-ERK, phospho-JNK, cyclin D1, CDK4, AMPK, PARP, caspase-3, Bcl-2, Bcl-xL, and Bax antibodies were purchased from Cell Signaling (Danvers, MA, USA). Anti-p38, ERK, JNK, GAPDH, and α-tubulin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Mun J.G., Jeon H.D., Yoon D.H., Lee Y.S., Park S.Y., Jin J.S., Park N.J, & Kee J.Y. (2022). Supercritical Extract of Cannabis sativa Inhibits Lung Metastasis in Colorectal Cancer Cells by Increasing AMPK and MAPKs-Mediated Apoptosis and Cell Cycle Arrest. Nutrients, 14(21), 4548.

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Lung Cancer Cell Line Culture and Antibodies

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Lung adenocarcinoma cell lines were cultured as described previously [22 (link)]. H1975, H2030, A549, H1650, and H441 were obtained from the American Type Culture Collection (ATCC). H2228 cells were a gift from J. Peter Koo (Yale). PC9, PC9/BRC1, H3255, and H3255 XLR were a gift from Katerina Politi (Yale). PC9/BRC1 and H3255 XLR cells are EGFR inhibitor resistant cells and have the EGFR T790M gatekeeper mutation [23 ].
Antibodies used include: PARP, phospho-p70S6K, p70S6K, phospho-AKT^Ser473, AKT, phospho-ERK, ERK, β-actin, caspase-8 (mouse), p105/p50, and p100/p52 (Cell Signaling); RIP1 (BD Pharmingen); caspase-8 (goat), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz); histone H3 (Abcam); c-FLIP (Enzo Life Sciences).

Langdon C.G., Wiedemann N., Held M.A., Mamillapalli R., Iyidogan P., Theodosakis N., Platt J.T., Levy F., Vuagniaux G., Wang S., Bosenberg M.W, & Stern D.F. (2015). SMAC mimetic Debio 1143 synergizes with taxanes, topoisomerase inhibitors and bromodomain inhibitors to impede growth of lung adenocarcinoma cells. Oncotarget, 6(35), 37410-37425.

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Immunoblotting Analysis of Signaling Pathways

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THP-1 macrophage-like cells, BMDM, HEK293T and A549 cells were lysed in ice-cold RIPA lysis buffer containing complete protease inhibitor and phosphatase inhibitor cocktails (Roche), resolved by SDS-PAGE, and transferred onto a PVDF membrane. The membranes were immunoblotted with antibodies against Phospho-NF-κB p65 (3033, Cell Signaling), Phospho-IκBα (9246, Cell Signaling), IκBα (4812, Cell Signaling), Phospho-ERK (4370, Cell Signaling), ERK (4695, Cell Signaling), Phospho-JNK (4668, Cell Signaling), Phospho-AKT (4060, Cell Signaling), AKT (9272, Cell Signaling), Phospho-STAT3 (9145, Cell Signaling), Flag®M2 (F1804, Sigma-Aldrich), SARS-CoV-2 S (GTX632605, GeneTex) and β-actin (A2228, Sigma). Immunoreactive protein bands were detected using ECL super signal west femto substrate reagent (Thermo Scientific).

Khan S., Shafiei M.S., Longoria C., Schoggins J., Savani R.C, & Zaki H. (2021). SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway. bioRxiv.

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Osteoclast Differentiation Signaling Pathways

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Recombinant M-CSF and RANKL were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies against phospho-JNK, JNK, phospho-ERK, ERK, phospho-p65, and phospho-IκBα were obtained from Cell Signaling Technology (Danvers, MA, USA). The antibody against NFATc1 was purchased from BD Pharmingen (San Diego, CA, USA). Fetal bovine serum (FBS) and α-minimum essential medium (α-MEM) were obtained from Gibco BRL (Grand Island, NY, USA).

Ihn H.J., Kim J.A., Lim S., Nam S.H., Hwang S.H., Lim J., Kim G.Y., Choi Y.H., Jeon Y.J., Lee B.J., Bae J.S., Kim Y.H, & Park E.K. (2019). Fermented Oyster Extract Prevents Ovariectomy-Induced Bone Loss and Suppresses Osteoclastogenesis. Nutrients, 11(6), 1392.

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Protein Extraction and Immunoblotting

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Proteins extraction and blotting was performed as in (24 (link)). The primary antibodies for phospho-ERK, total-ERK, phospho-Histone 3, Mcl-1, BIM and total MEK, were from Cell Signaling Technology. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Sigma Aldrich) was used as a loading control to demonstrate even protein loading. The secondary antibodies; goat anti-rabbit IgG HRP and sheep anti-mouse IgG HRP were from Amersham/ GE Healthcare. For mouse xenograft experiments, tumor samples were harvested and immediately stored in RNAlater solution (Ambion) at 4°C before protein extraction.

Phadke M.S., Sini P, & Smalley K.S. (2015). The novel ATP-competitive MEK/Aurora kinase inhibitor BI-847325 overcomes acquired BRAF inhibitor resistance through suppression of Mcl-1 and MEK expression. Molecular cancer therapeutics, 14(6), 1354-1364.

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