Phosphorylated erk

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Phosphorylated ERK is a laboratory tool used to detect and quantify the phosphorylation state of the extracellular signal-regulated kinase (ERK) protein. ERK is a key component of the mitogen-activated protein kinase (MAPK) signaling pathway, which plays a crucial role in various cellular processes, such as cell growth, proliferation, and differentiation. The phosphorylation of ERK is a common method for evaluating the activation of this signaling pathway.

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57 protocols using phosphorylated erk

Antibody Sources for Protein Analysis

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Antibodies used in this study were obtained from the following sources: α-tubulin (Santa Cruz Biotechnology, CA, USA): Hemagglutinin (HA) (Roche); Erk, and phosphorylated Erk (Cell Signaling Technology, MA, USA). Recombinant SEMA3E was purchased from R&D Systems (R&D Systems, MN, USA).

Maejima R., Tamai K., Shiroki T., Yokoyama M., Shibuya R., Nakamura M., Yamaguchi K., Abue M., Oikawa T., Noguchi T., Miura K., Fujiya T., Sato I., Iijima K., Shimosegawa T., Tanaka N, & Satoh K. (2016). Enhanced expression of semaphorin 3E is involved in the gastric cancer development. International Journal of Oncology, 49(3), 887-894.

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Molecular Pathway Regulation in Cells

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The primary antibodies against proliferating cell nuclear antigen (PCNA), phosphorylated histone H3 (P-H3) and GAPDH were obtained from Santa Cruz Biotechnology. The antibodies of phosphorylated Akt, phosphorylated ERK, Akt, ERK cyclin E, cyclin D1, p21 and p27 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). The ZEB1 and ZNF217 antibodies were purchased from Abcam (Cambridge, UK). LY294002 were obtained from Selleck Chemicals (Houston, TX, USA). U0126 was purchased from Beyotime Biotechnology (Shanghai, China). The siRNA specifically targeting lncRNA-ATB and non-targeting control were synthesized by GenePharma Co. (Shanghai, China).

XU S., YI X.M., TANG C.P., GE J.P., ZHANG Z.Y, & ZHOU W.Q. (2016). Long non-coding RNA ATB promotes growth and epithelial-mesenchymal transition and predicts poor prognosis in human prostate carcinoma. Oncology Reports, 36(1), 10-22.

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Western Blot Protein Expression Analysis

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A standard western blot assay was used to analyse protein expression, using primary antibodies against signal transducer and activator of transcription 3 (STAT3) (1 : 1000; Cell Signaling Technology, Beverly, Massachusetts, USA), phosphorylated STAT3 (1 : 1000; Cell Signaling Technology), extracellular signal‐regulated kinase (ERK) 1 (1 : 1000; Santa Cruz Biotechnology, Santa Cruz, USA), phosphorylated ERK (1 : 2000; Cell Signaling Technology), PCNA (1 : 1000; Cell Signaling Technology) and β‐tubulin (1 : 5000; Sigma Aldrich), as described previously²⁰. The immunoreactive signals were visualized by scanning densitometry with the ChemiDoc™ Touch Imaging System (Bio‐Rad Laboratories, Hercules, California, USA).

Shi J.H., Yan X., Zhang S.J, & Line P.D. (2020). Simulated model of RAPID concept: highlighting innate inflammation and liver regeneration. BJS Open, 4(5), 893-903.

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Colorectal and Lung Cancer Cell Culture and Targeting HGF

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The human colorectal cancer cell lines, HCT116, HT29 and DLD-1 and lung cancer cell line 226Br were cultured in RPMI1640 (Welgene, Seoul, Korea) containing 10% fetal bovine serum (FBS; Welgene) and antibiotics (100 mg/L penicillin and 100 mg/L streptomycin; GIBCO-BRL Life Technologies; Gaithersburg, MD, USA). Human colonic fibroblasts (CCD-18co) were cultured in DMEM (Welgene, Seoul, Korea) containing 10 % FBS and antibiotics Cells were incubated in a humidified thermostat under 5 % CO₂ at 37 °C. A predesigned siRNA targeting HGF and scramble siRNA were purchased from Shanghai Genepharma (Shanghai, China). Recombinant Human HGF was purchased from R&D system (Minneapolis, MN, USA). Humanized anti-HGF antibody was kindly provided by YooYoung pharmaceutical Co. Ltd (Seoul, Korea) and iBIO Inc (Seoul, Korea). Irinotecan (CPT-11) was obtained from Sigma Aldrich (St. Louis, MO, USA). Antibodies to c-MET, phosphorylated-c-MET, phosphorylated-ERK, phosphorylated-AKT, PARP, and cleaved Caspase-3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody against HGF, actin and fibroblast marker (ER-TR7) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Woo J.K., Kang J.H., Kim B., Park B.H., Shin K.J., Song S.W., Kim J.J., Kim H.M., Lee S.J, & Oh S.H. (2015). Humanized anti-hepatocyte growth factor (HGF) antibody suppresses innate irinotecan (CPT-11) resistance induced by fibroblast-derived HGF. Oncotarget, 6(27), 24047-24060.

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Evaluation of Cancer Cell Responses

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Dulbecco’s Modified Eagle’s Medium (DMEM) medium, Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), penicillin/streptomycin, L-glutamine, phosphate-buffered saline (PBS) and trypsin-EDTA were obtained from Gibco (Grand Island, NY, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), dimethyl sulfoxide (DMSO), Hoechst33342, propidium iodide (PI), cisplatin, doxorubicin and bovine serum albumin (BSA) were obtained from Sigma-Aldrich, Co. (St. Louis, MO, USA). The following primary antibodies, caspase-9 (#9502), caspase-3 (#9662), p53 (#9282), BCL-2 (#4223), BAX (#5023), AKT (#9272), phosphorylated AKT (#4060), ERK (#4695), phosphorylated ERK (#4370), Nanog (#4903), CD44 (#9502), GADPH (#5174) and β-actin (#4970), were obtained from Cell Signaling Technology (Danvers, MA, USA). CD133 (#CA1217) was obtained from Cell Applications (San Diego, CA, USA). The respective secondary antibodies, anti-rabbit IgG (#7074) and anti-mouse (#7076), were obtained from Cell Signaling Technology (Danvers, MA, USA).

Chantarawong W., Chamni S., Suwanborirux K., Saito N, & Chanvorachote P. (2019). 5-O-Acetyl-Renieramycin T from Blue Sponge Xestospongia sp. Induces Lung Cancer Stem Cell Apoptosis. Marine Drugs, 17(2), 109.

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Protein Extraction and Western Blot Analysis

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Protein from cells and tissue was extracted using Passive Lysis Buffer (Promega) with protease/phosphatase inhibitor mini tablets, EDTA free (Pierce). Cells were resuspended and rocked in lysis buffer for 15 minutes, centrifuged at maximum speed for 1 minute. Supernatants were then collected. Frozen tissue was homogenized in lysis buffer using Bullet Blender Bead Lysis tubes (MidSci) and a Bullet Blender Storm 24 (Next Advance) set at Speed 10 for approximately 10 minutes. Homogenates were centrifuged at 4°C at maximum speed for 1 minute, and supernatants were collected. Protein concentration was determined using the BCA assay (Pierce). Cell and tissue protein extracts (35–40μg protein/lane) were loaded onto a 4–20% SDS-gel (BioRad). Proteins were transferred onto PVDF membrane (Millipore). Membranes were incubated in blocking solution (5% milk in TBST). Membranes were incubated with primary antibodies: THM1 (Sigma), phosphorylated AKT (Cell Signaling), phosphorylated ERK (Cell Signaling), AKT (Cell Signaling), ERK (Cell Signaling), ß-Actin (Cell Signaling), PPARγ (Cell Signaling), and CEBPα (Cell Signaling) overnight at 4°C. Membranes were washed and incubated with secondary antibodies conjugated to HRP (Cell Signaling). Signal was detected using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) and imaged using an Amersham Imager 600.

Jacobs D.T., Allard B.A., Pottorf T.S., Silva L.M., Wang W., Al-Naamani A., Agborbesong E., Wang T., Carr D.A, & Tran P.V. (2019). Intraflagellar-transport A dysfunction causes hyperphagia-induced systemic insulin resistance in a pre-obese state. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(1), 148-160.

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Macrophage Inflammatory Signaling Modulation

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RAW 264.7 macrophages were plated in 6-well plates (4 × 10⁵/well) and cultured in 2 mL of DMEM for 4 h. The cultures were washed to remove non-adherent cells and then incubated with 2 mL of complete DMEM for 20 h. The culture medium was replaced with DMEM for 30 min to allow the cells to adjust. (i) To induce an inflammation model, 1 μg/mL LPS (Sigma) was added. After 24 h of stimulation with LPS, the cells were treated with the indicated concentrations of L-4F (0, 0.1, or 0.25 μg/mL) for 12 h, and the levels of pp38, pJNK and pERK were analyzed. (ii) Cells were stimulated by LPS and treated with the indicated concentrations of L-4F (0, 0.1, or 0.25 μg/mL) for 1 h, and the levels of pSTAT3 were analyzed.
Western blot procedure: Briefly, cells were lysed in radio-immunoprecipitation assay buffer containing the phosphatase and protease inhibitors phenylmethanesulfonyl fluoride and aprotinin (Sigma), and protein was collected. Then, the protein was separated by SDS-PAGE, transferred to PVDF membranes (Roche) and probed with the indicated primary antibodies (phosphorylated STAT3, phosphorylated p38, phosphorylated JNK and phosphorylated ERK; Cell Signaling Technology). The antibody-antigen complexes were detected using a Chemiluminescent HRP substrate kit (Millipore) according to the manufacturer’s protocols.

Peng M., Zhang Q., Cheng Y., Fu S., Yang H., Guo X., Zhang J., Wang L., Zhang L., Xue Z., Li Y., Da Y., Yao Z., Qiao L, & Zhang R. (2017). Apolipoprotein A-I mimetic peptide 4F suppresses tumor-associated macrophages and pancreatic cancer progression. Oncotarget, 8(59), 99693-99706.

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Western Blot Analysis of Protein Expression

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Cell pellets were lysed in RIPA buffer containing 10 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 1% DOC, 0.1% SDS (supplemented with 1 µg/ml aprotinin, 2 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 mM DTT, and 0.1 M PMSF) for 20 minutes on ice and centrifuged at high speed for 20 minutes at 4 °C. Extracts containing equal quantities of proteins, determined by using the BCA Protein Assay Kit (Pierce) were separated by SDS-polyacrylamide gel electrophoresis (12.5% acrylamide) and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were incubated in blocking buffer [5% (w/v) non-fat dry milk dissolved in PBST (1X PBS containing 0.05% [v/v] Tween 20)] for 1 hour at room temperature. Membranes were then incubated with antibodies to phosphorylated ERK (Cell Signaling), E-Cadherin (Cell Signaling), ACSL4 (Thermo), or Actin (Abcam) diluted 1:1000 in blocking buffer for 1–2 hours at room temperature. Secondary antibodies conjugated to horse-radish peroxidase were obtained from Biorad and used at a dilution of 1:10,000. Bound antibodies were detected using enhanced chemiluminescence (Biorad).

Taylor W.R., Fedorka S.R., Gad I., Shah R., Alqahtani H.D., Koranne R., Kuganesan N., Dlamini S., Rogers T., Al-Hamashi A., Kholodovych V., Barudi Y., Junk D., Rashid M.S., Jackson M.W, & Tillekeratne L.M. (2019). Small-Molecule Ferroptotic Agents with Potential to Selectively Target Cancer Stem Cells. Scientific Reports, 9, 5926.

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Melanogenesis Regulation Mechanism Study

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LQ and LQG were purchased from Wako Pure Chemical Industries (Osaka, Japan) and Tokiwa Phytochemical (Tokyo, Japan), respectively. α-Melanocyte-stimulating hormone (α-MSH) was purchased from Sigma Chemical (St. Louis, MO, USA). Antibodies against tyrosinase, TRP-1, TRP-2, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against MITF, phosphorylated CREB, CREB, phosphorylated Akt, Akt, phosphorylated ERK, ERK, phosphorylated p38, and p38 were obtained from Cell Signaling Technology (Beverly, MA, USA). Fetal bovine serum (FBS) was supplied by GIBCO (Gaithersburg, MD, USA). H-89 was purchased from AdipoGen (San Diego, CA, USA). All other chemicals were obtained from Wako Pure Chemical Industries.

Uto T., Ohta T., Yamashita A., Fujii S, & Shoyama Y. (2019). Liquiritin and Liquiritigenin Induce Melanogenesis via Enhancement of p38 and PKA Signaling Pathways. Medicines, 6(2), 68.

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Western Blot Analysis of Inflammatory Markers

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Western blot analysis was done, as explained below [16 (link),22 (link)]. The primary antibodies were treated during 24 hours for TNF-α (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-1β (1:1,000; Santa Cruz Biotechnology), NF-κB (1:1,000; Abcam, Cambridge, UK), IκBα (1:1,000; Santa Cruz Biotechnology), phosphorylated IκBα (p-IκBα) (1:1,000; Santa Cruz Biotechnology), extracellular signal-regulated kinase (ERK) (1:1,000; Cell Signaling Technology), phosphorylated ERK (1:1,000; Cell Signaling Technology), c-Jun N-terminal kinases (JNK) (1:1,000; Cell Signaling Technology), phosphorylated JNK (1:1,000; Cell Signaling Technology), p38 (1:1,000; Cell Signaling Technology), and phosphorylated p38 (1:1,000; Cell Signaling Technology). The secondary antibodies were applied to the membrane, and then the bands were calculated by enhanced chemiluminescent kit.

Kim S.H., Ko Y.J., Kim J.Y, & Sim Y.J. (2021). Treadmill Running Improves Spatial Learning Memory Through Inactivation of Nuclear Factor Kappa B/Mitogen-Activated Protein Kinase Signaling Pathway in Amyloid-β-Induced Alzheimer Disease Rats. International Neurourology Journal, 25(Suppl 1), S35-43.

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