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Erk1 2

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ERK1/2 is a protein that plays a central role in the regulation of cell growth and differentiation. It is a member of the mitogen-activated protein kinase (MAPK) family and is involved in the transmission of signals from the cell surface to the nucleus.

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Lab products found in correlation

Elisa kit, by Cell Signaling Technology (2 mentions) β actin, by Merck Group (2 mentions) P akt ser473, by Cell Signaling Technology (2 mentions) Phospho erk1 t202 y204 erk2 t185 y187, by R&D Systems (1 mentions) Phospho stat3 tyr705, by Cell Signaling Technology (1 mentions) Gapdh, by Santa Cruz Biotechnology (1 mentions) Survivin, by Santa Cruz Biotechnology (1 mentions) Actin, by Santa Cruz Biotechnology (1 mentions) Ldc1267, by Selleck Chemicals (1 mentions) Cip2a, by Santa Cruz Biotechnology (1 mentions) P axl y779, by R&D Systems (1 mentions) Anti pp2a, by Cell Signaling Technology (1 mentions) Biotinylated secondary antibody, by Vector Laboratories (1 mentions) Diphenyleneiodonium dpi, by Merck Group (1 mentions) Iba1 antibody, by Abcam (1 mentions) Anti p38, by R&D Systems (1 mentions) Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions) Vectastain abc kit, by Vector Laboratories (1 mentions) Horseradish peroxidase conjugated secondary antibody, by Cytiva (1 mentions) Enhanced chemiluminescence western blotting detection reagent, by Cytiva (1 mentions)

9 protocols using erk1 2

1

Quantifying MAPK/SAPK Activation and Inflammatory Markers

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The activation of p38 mitogen-activated protein kinase/stress-activated protein kinase (MAPK/SAPK) is determined by the quantity of p38 MAPK/SAPK that is phosphorylated in the pathway. The level of phosphorylation was estimated using an ELISA kit (Cell Signaling Technology, Danvers, MA, USA). In addition, the expression of IL-1β, IL-6, and TNF-α in the cell culture supernatants and the total and phosphorylated levels of the extracellular signal-regulated kinase (ERK) 1/2 (R&D Systems, Minneapolis, MN, USA) and p65 subunit of nuclear factor kappa B (NF-κB) (Cell Signaling Technology, Danvers, MA, USA) in the nuclear lysates were detected using appropriate ELISA kits.
Kim N., Kim C., Ryu S.H., Lee W, & Bae J.S. (2022). Anti-Septic Functions of Cornuside against HMGB1-Mediated Severe Inflammatory Responses. International Journal of Molecular Sciences, 23(4), 2065.
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2

NGF and GM-CSF Stimulation Assay

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Cell lines were stimulated with 100 ng/mL recombinant NGF or 2 ng/mL recombinant GM-CSF and incubated at 37°C for 15 minutes before being lysed using HEPES lysis buffer (1% triton) with phosphatases inhibitory cocktail (SER-TRE Cat No. P2850 and TYR Cat No. P2850; Sigma-Aldrich). Some groups were treated with varying concentrations of the TRK inhibitor, entrectinib, for 2 hours prior to NGF addition. SDS-PAGE, PVDF transfer, and Western immunoblot were all performed using standard techniques (BioRad Manual). Staining antibodies for western immunoblotting included total anti-TRKA (Cat No. ab1445; Abcam) and anti-phosphorylated TRKA-Tyr490 (Cat No. ab37837; Abcam), ERK1/2 (Cat No. AF1576; R&D Systems), p-ERK1/2 Tyr202/Tyr204 (Cat No. AF1018; R&D Systems), and b-actin. Protein bands were quantified using ImageJ for statistical analysis [45 (link)].
Herbrich S.M., Kannan S., Nolo R.M., Hornbaker M., Chandra J, & Zweidler-McKay P.A. (2018). Characterization of TRKA signaling in acute myeloid leukemia. Oncotarget, 9(53), 30092-30105.
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3

Western Blot Analysis of Sunitinib Targets

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For Western blot analysis, 15 to 20 µg of total cell lysates were diluted in 15 µl of Laemmli buffer, separated on a 10% SDS-PAGE gel and transferred to nitrocellulose membranes by semi-dry blotting. The membranes were blocked with 5% milk powder in 0.02% Tween TBS (TBST) for 1 hour. Based on previous results on key molecules regulated by Sunitinib (Pfizer, investigators brochure, March 2008), membranes were incubated with antibodies against Akt, phospho-Akt (Ser473), STAT3, Phospho-STAT3 (Tyr705) (all from Cell Signaling, Danvers, USA), ERK1/2, phospho-ERK1 (T202/Y204)/ERK2 (T185/Y187) (all from R&D Systems, Minneapolis, USA), and β-Actin (Sigma Aldrich, Missouri, USA) or GAPDH (Santa Cruz Biotechnology, Heidelberg, Germany) overnight at 4°C. Bound antibodies were visualized with a horseradish peroxidase–linked antibody against mouse or antibody against rabbit immunoglobulin G (R&D Systems, Minneapolise, USA) followed by enhanced chemiluminescence reaction (Roche Applied Science, Basel, Switzerland). Western blots were repeated three times.
Moeckel S., Meyer K., Leukel P., Heudorfer F., Seliger C., Stangl C., Bogdahn U., Proescholdt M., Brawanski A., Vollmann-Zwerenz A., Riemenschneider M.J., Bosserhoff A.K., Spang R, & Hau P. (2014). Response-Predictive Gene Expression Profiling of Glioma Progenitor Cells In Vitro. PLoS ONE, 9(9), e108632.
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4

AXL-Fc Receptor Inhibition Assay

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AXL-Fc protein fragment (AXL-Fc) was purchased from R&D Systems (St. Paul, MN, USA). LDC1267 (a TAM family inhibitor) was purchased from Selleck (Houston, TX, USA). Rat GAS6 recombinant protein (RPA204Ra01) was purchased from Cloud-clone Corp. Antibodies for immunoblotting, including DAPK, p-DAPK (S308), CIP2A, AKT, MYC, Survivin and Actin, were purchased from Santa Cruz Biotechnology (San Diego, CA). ERK1/2, p-ERK1 (T202/Y204) / ERK2 (T185/Y187), and p-AXL (Y779) were purchased from R&D Systems (St. Paul, MN, USA). Other antibodies, including anti-PP2A and p-AKT (Ser473), were purchased from Cell Signaling (Danvers, MA).
Chen Y.L., Tsai Y.T., Chao T.T., Wu Y.N., Chen M.C., Lin Y.H., Liao C.H., Chou S.S, & Chiang H.S. (2018). DAPK and CIP2A are involved in GAS6/AXL-mediated Schwann cell proliferation in a rat model of bilateral cavernous nerve injury. Oncotarget, 9(5), 6402-6415.
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5

Neuroinflammation and Oxidative Stress

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Materials relating cell cultures were from Invitrogen(Carlsbad, CA, USA). [3H]dopamine (DA) (28Ci/mmol) and [3H] gamma-aminobutyric acid (GABA) (81Ci/mmol) were from Perkin Elmer Life Science (Boston, MA, USA). Polyclonal anti-tyrosine hydroxylase (TH), anti-complement receptors 3 (OX-42), anti-neuron-specific neuclear protein (Neu N), anti-protein kinase C-σ (PKC-σ), anti-P38, anti-extracellular signal-regulated kinase1/2 (ERK1/2), anti-c-Jun N-terminal kinase (JNK) and anti-nuclear factor-КBP50 (NF-КB P50) antibodies were from R&D Systems (Minneapolis, MN, USA). Anti-ionized calcium binding adaptor molecule-1 (Iba-1) antibody was from Abcam (330 Cambridge Science Park, Cambridge, UK). Vectastain ABC kit and biotinylated secondary antibodies were from Vector Laboratories (Burlingame, CA, USA); Fluorescence probe 2’,7’-dichlorodi-hydrofluorescein (DCFH-DA), cytosine arabinoside (Ara-C), Leu-Leu methyl ester hydrobromedia (LME), superoxide dismutase (SOD), 2-(4 lodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazo1ium (WST-1) and diphenyleneiodonium (DPI) were from Sigma-Aldrich (St. Louis, MO, USA). SYBR green PCR master mix was from Applied Biosystems (Foster City, CA, USA).
Zhang W., Gao J.H., Yan Z.F., Huang X.Y., Guo P., Sun L., Liu Z., Hu Y., Zuo L.J., Yu S.Y., Cao C.J., Wang X.M, & Hong J.S. (2016). Minimally toxic dose of lipopolysaccharide and α-Synuclein oligomer elicit synergistic dopaminergic neurodegeneration: role and mechanism of microglial NOX2 activation. Molecular neurobiology, 55(1), 619-632.
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6

Western Blot Analysis of Signaling Proteins

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Western blotting analyses were performed as previously described [30 (link)]. Protein samples containing 8 μg of total protein were separated via electrophoresis with 4–15% sodium dodecyl sulfate (SDS)-polyacrylamide gels, after which they were transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). After transfer, PVDF membranes were blocked with 5% bovine serum albumin (Wako Pure Chemical Industries, Osaka, Japan) in TBS containing 0.1% Tween-20 at room temperature for 1 h.
Immunoblotting was then performed using primary antibodies against phospho-Akt (p-Akt) (Ser473; 1:1000, Cell Signaling Technology, Danvers, MA, USA), Akt (1:1000, Cell Signaling Technology), phospho-ERK1(T202/Y204)/ERK2(T185/Y187) (p-ERK1/2) (1:2000, R&D Systems, Minneapolis, MN, USA), and ERK1/2 (1:2000, R&D Systems). Horseradish peroxidase-conjugated secondary antibody was used to detect immunoreactivity (Amersham Pharmacia Biotech, Piscataway, NJ, USA), which was visualized using enhanced chemiluminescence western blotting detection reagents (Amersham Pharmacia Biotech) and RX-U Fuji X-ray film (Fuji Film, Tokyo, Japan). Data were analyzed using ImageJ software.
Terada K., Migita K., Matsushima Y., Sugimoto Y., Kamei C., Matsumoto T., Mori M., Matsunaga K., Takata J, & Karube Y. (2018). Cholinesterase inhibitor rivastigmine enhances nerve growth factor-induced neurite outgrowth in PC12 cells via sigma-1 and sigma-2 receptors. PLoS ONE, 13(12), e0209250.
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7

Evaluating Inflammatory Signaling Pathways

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The level of activation of p38 mitogen-activated protein kinase/stress-activated protein kinase (MAPK/SAPK) was determined by measuring the amount of phosphorylated p38 MAPK/SAPK using an ELISA kit (Cell Signaling Technology, Danvers, MA, USA). The expression levels of IL-1β, IL-6, and TNF-α in the cell culture supernatants, as well as the total and phosphorylated levels of extracellular signal-regulated kinase (ERK) 1/2 (R&D Systems, Minneapolis, MN, USA) and the p65 subunit of nuclear factor kappa B (NF-κB) in the nuclear lysates, were measured using ELISA kits.
Park Y.J., Heo J.B., Choi Y.J., Cho S., Lee T., Song G.Y, & Bae J.S. (2024). Antiseptic Functions of CGK012 against HMGB1-Mediated Septic Responses. International Journal of Molecular Sciences, 25(5), 2976.
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8

Western Blot Analysis of Protein Targets

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Amount of 5 μg extracted protein was used for Western blot analysis. Specific primary antibodies were used to probe the target protein: Antibody for TSLP (Abcam, Cambridge, UK, ab47943 and Sigma, St. Louis, Mo, USA, PRS4023), phosphorylated-ERK1/2, ERK1/2, phosphorylated AKT1/2, AKT1/2, phosphorylated-Src, Src, phosphorylated GSK3α/β, GSK3α/β, phosphorylated STAT2, STAT2, phosphorylated p53, p53 (all from R&D Systems), phosphorylated AMPKα1 (Cell Signalling), AMPKα1 (Cell Signalling), EFNB2 (Abcam), PBX1 (Abcam) or β-actin (Sigma-Aldrich). After probing with HRP-linked secondary antibody (GE Healthcare, Chicago, IL, USA), the membrane was detected with ECL reagent (PerkinElmer, Boston, MA, USA).
Chan L.K., Lau T.S., Chung K.Y., Tam C., Cheung T.H., Yim S.F., Lee J.H., Leung R.W., Qin J., Or Y.Y., Lo K.W, & Kwong J. (2021). Short-Form Thymic Stromal Lymphopoietin (sfTSLP) Is the Predominant Isoform Expressed by Gynaecologic Cancers and Promotes Tumour Growth. Cancers, 13(5), 980.
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9

Protein Expression and Signaling Analysis

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The following antibodies and reagents were purchased from the sources indicated: ERK 1/2 (R&D Cat# 9107S); pERK 1/2 (R&D Cat# 9101S); CDX2 (LSbio Cat# LS-B9317); NFĸB (Abcam cat # 16502), pNFĸB p65 (Cell Signaling Cat # 3031S); Claudin 1 (Invitrogen Cat# 51-9000); Claudin 2 (Invitrogen Cat# 32-5600); Claudin 3 (Invitrogen Cat# 341700); Claudin 7 (Invitrogen Cat# 374800); goat anti-rabbit-HRP (Santa Cruz Biotechnology Cat# sc-2004); goat anti-mouse-HRP (Santa Cruz Biotechnology Cat# sc-2005) and; actin (Sigma-Aldrich Cat# A1978). U0126 (Cat # 19-147) was from Sigma-Aldrich.
Chivero E.T., Ahmad R., Thangaraj A., Periyasamy P., Kumar B., Kroeger E., Feng D., Guo M.L., Roy S., Dhawan P., Singh A.B, & Buch S. (2019). Cocaine Induces Inflammatory Gut Milieu by Compromising the Mucosal Barrier Integrity and Altering the Gut Microbiota Colonization. Scientific Reports, 9, 12187.
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