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Amphiregulin

Amphiregulin is a member of the epidermal growth factor (EGF) family of proteins.
It is involved in cell proliferation, differentiation, and survival.
Amphiregulin binds to and activates the EGF receptor, triggering intracellular signaling cascades that regulate various cellular processes.
It plays a role in the development and homeostasis of multiple tissues, including the skin, intestine, and mammary gland.
Dysregulation of Amphiregulin has been implicated in the pathogenesis of certain cancers and inflammatory disorders.
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Most cited protocols related to «Amphiregulin»

Plasmids encoding AP-tagged EGFR ligands were constructed by inserting partial cDNAs for human TGFα, amphiregulin, epiregulin, EGF, betacellulin, and HB-EGF into the 3′ end of human placental AP cDNA on a pRc/CMV-based expression vector pAlPh. pAlPh contains an NH2-terminally located HB-EGF signal sequence. In all cases, the junction between AP and the EGFR ligand was placed next to the membrane-proximal EGF repeat. Release of the AP module into the culture supernatant thus requires cleavage at the COOH-terminal cleavage site. It should be noted that the results obtained with AP-tagged EGFR ligands corroborate previous results that TGFα, HB-EGF, and amphiregulin are cleaved by ADAM17 (Peschon et al., 1998 (link); Merlos-Suarez et al., 2001 (link); Sunnarborg et al., 2002 (link); Jackson et al., 2003 (link)). This validates the use of AP tags to measure ectodomain shedding for these substrates. In addition, the use of an AP tag has been independently validated using the TNF family members TNFα and TRANCE/OPGL (Zheng et al., 2002 (link); Chesneau et al., 2003 (link)). This strongly suggests that an AP tag, which provides a sensitive and quantitative means of measuring ectodomain shedding, should not interfere with the shedding properties of the other EGFR ligands tested here.
Publication 2004
ADAM17 protein, human Amphiregulin Betacellulin Cloning Vectors Cytokinesis DNA, Complementary EGFR protein, human Epiregulin Family Member Heparin-binding EGF-like Growth-Factor Homo sapiens Ligands Placenta Plasmids Signal Peptides Tissue, Membrane TNFSF11 protein, human Transforming Growth Factor alpha
CD271low/MUC1high primary luminal epithelial cells were seeded at 6000 cells/cm2 on confluent fibroblast feeders of CD105high/CD26low and CD105low/CD26high cells, respectively, in modified breastoid base medium without HEPES [9 (link)] (DMEM/F-12, 1:1), 1 μg/ml hydrocortisone (Sigma-Aldrich), 9 μg/ml insulin (Sigma-Aldrich), 5 μg/ml transferrin (Sigma-Aldrich), 5.2 ng/ml Na-Selenite (BD Industries), 100 μM ethanolamine (Sigma-Aldrich), 20 ng/ml basic fibroblast growth factor (PeproTech), 5 nM amphiregulin (R&D Systems), with the addition of 10 μM Y-27632 (Axon Medchem), 1.8 × 10−4 M adenine (Sigma Aldrich) and the serum replacement B27 (20 μl/ml, Life Technologies) [6 (link)]. hMSC feeders cultured under similar conditions were used for comparison.
To determine whether luminal progenitors and differentiated cells responded differently to co-culture, FACS-sorted CD166 (ALCAM)high/laminin receptor 67LRhigh (EpCAMhigh/CD166high/LNR67high) or CD166high (EpCAMhigh/CD90low/CD166high) differentiated luminal cells versus 67LRlow (EpCAMhigh/CD166low/LNR67low) or CD166low (EpCAMhigh/CD90low/CD166low) progenitors [6 (link)] were confronted with either CD105high/CD26low or CD105low/CD26high cells under similar conditions. Epithelial structure formation was observed for up to three weeks by phase contrast microscopy and photographed (Leica DM IL).
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Publication 2016
Activated-Leukocyte Cell Adhesion Molecule Adenine Amphiregulin Axon Cells Coculture Techniques Epithelial Cells Ethanolamine Fibroblast Growth Factor 2 Fibroblasts HEPES Hydrocortisone Insulin Laminin Receptor Microscopy, Phase-Contrast Phenobarbital Selenite Serum Transferrin Y 27632

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Publication 2019
alpha HML-1 Amphiregulin anti-IgM Antibodies Antibodies, Anti-Idiotypic Antigen T Cell Receptor, beta Chain Cardiac Arrest CD44 protein, human Cells Collagenase Cytokine Deoxyribonucleases Euthanasia Flow Cytometry gamma-delta T-Cell Receptor Goat Hematopoietic System Hybridomas IL17A protein, human Immunoglobulins Interferon Type II Interleukin-17F ITGAM protein, human Lung MAG protein, human Mucolipidosis Type IV Mus Nodes, Lymph Protein Transport Protoplasm RORC protein, human Spleen Tissues Transcription Factor Tumor Necrosis Factor-alpha
Fibrillar collagen I, the most abundant structural protein in mammary glands, plays an important role in normal development as well as in breast cancer. Our previous studies have demonstrated that collagen I induces a conserved response of protrusive invasion in both normal and tumor organoids [23 (link)]. This section describes how to properly prepare preassembled collagen I and embed mammary organoids in a 3D gel.

Use a plate with a glass bottom for time-lapse imaging.

Use 20-30 μL of neutralized collagen to make a thin underlay on the cover glass of the well at room temperature (Fig. 5e). The underlay helps the top collagen/organoid suspension attach better to the cover glass.

Incubate the plate with the underlays at 37 °C until ready for plating.

Preincubate the neutralized collagen I solution (used for the top gel) at 4 °C for 60–120 min for preassembly [24 (link)] (seeNote 14). The collagen I solution will turn cloudy and fibrous (Fig. 5d1–6) (seeNote 15), a state we term preassembled collagen I.

Set up the tissue culture hood in preparation for plating (Fig. 4a).

Set the heating block to 37 °C, and place the plate on top, in direct contact with the block (Fig. 4b–c′) (seeNote 10).

Always keep the collagen I solution on ice. Add the desired amount of preassembled collagen I to the organoid pellet in a microcentrifuge tube. Since collagen I is quite viscous, first pipette up and down slowly a few times to coat the tip and ensure an accurate volume.

Keep the tube on ice or in a cold block. Resuspend the organoid pellet gently to avoid introducing air bubbles. Do not try to take up the entire volume into the pipette tip while mixing.

Plate the appropriate volume of collagen/organoid suspension (see table in Subheading 3.4) on top of the underlay (Fig. 5e′). Pipette up and down to resuspend the organoids before plating each sample, and pipette out only until the first stop.

Keep the plate on the heating block for several minutes to allow further gelation before returning it to the incubator (seeNote 16).

Incubate the plate at 37 °C, 5 % CO2, for 45–60 min. After gelation, collagen I fibrils are visible under the microscope at 10× and 40× (Fig. 5f-f′).

Gently add pre-warmed organoid medium supplemented with growth factor to the wells. A variety of growth factors may be used, including EGF ligands (EGF, TGF-α, amphiregulin, heregulin, neuregulin), FGF ligands (FGF2, FGF7), and HGF. We most commonly use 2.5 nM FGF2.

Add sterile water or PBS to the empty wells to prevent desiccation.

Label the wells. Return the plate to the incubator.

If the plate will be used for DIC imaging, use a glass plate cover for better image quality.

Publication 2015
Amphiregulin Breast Breast Carcinoma Cold Temperature Collagen Collagen Type I Desiccation FGF7 protein, human Fibroblast Growth Factor 2 Fibrosis Growth Factor Ligands Mammary Gland Microscopy Neoplasms Neuregulins Organoids Proteins Sterility, Reproductive TGFA protein, human Tissues Viscosity
MET and EGFR gene copy numbers were evaluated by FISH; a CEP7 centromere probe (Abbott Molecular) was used as a control. High-level MET amplification was defined as tight gene clusters of ≥ 15 copies in ≥ 10% of tumor cells, or a MET: CEP7 ratio of ≥2. A cutoff of ≥5 copies of MET/ cell was predefined as the criterion for FISH-positive status (FISH+), based on prior prognostic data supporting this cutoff in NSCLC (4 (link)). Tumors were considered EGFR FISH+ based on a scoring system used in multiple clinical studies (19 (link)). Further details are included in the Supplementary Methods section.
DNA and RNA were isolated from macrodissected tissue to enrich for tumor content. EGFR and KRAS mutations were evaluated using the DxS Genotyping Kit. MET exon 14 variants were evaluated by Surveyor nuclease digestion and detection by WAVE analysis (Transgenomics, Inc.). A polymorphism at position N375S of MET was evaluated by pyrosequencing on the Pyromark Q24 (11 (link)). Expression of MET, HGF, EGFR, amphiregulin (AREG), and epiregulin (EREG) mRNA transcripts was evaluated by qRT-PCR on the Biomark platform (Fluidigm). The primer/probes used for profiling are shown in Supplementary Table S2 and further details are included in the Supplementary Methods section.
Publication 2014
Amphiregulin Cells Centromere Digestion EGFR protein, human Epiregulin Exons Fishes Genes Genetic Polymorphism K-ras Genes Mutation Neoplasms Non-Small Cell Lung Carcinoma Oligonucleotide Primers RNA, Messenger Tissues

Most recents protocols related to «Amphiregulin»

RNA from GCM was extracted using TRIzol (Invitrogen) and purified with PureLink RNA columns (Life Technologies). RNA samples were treated with DNase I, and reverse transcription was performed with High-Capacity cDNA Reverse Transcription Kit (Life Technologies). Real-time PCR was performed using the TaqMan Gene expression assay (Applied Biosystems) following the manufacturer’s instructions, on cDNA specimens in triplicate, using 1× Universal PCR Master Mix (Life Technologies) and 1× mix containing specific receptors probes (Life Technologies). Relative quantification was calculated from the ratio between the cycle number (Ct) at which the signal crossed a threshold set within the logarithmic phase of the given gene and that of the reference β-actin gene (4310881E; Life Technologies). Mean values of the triplicate results for each animal were used as individual data for 2-ΔCt statistical analysis. The following is the list of probes used: nicotinic cholinergic receptor, gamma subunit (AChRγ) (CHRNG; Mm00437419_m1; Life Technologies), epsilon subunit (AChRε) (CHRNE; Mm00437411_m1; Life Technologies), alpha subunit (AChRα1) (CHRNA1; Mm00431629_m1; Life Technologies), beta subunit (AChRβ1) (CHRNB1; Mm00680412_m1; Life Technologies), delta subunit (AChRδ) (CHRND; Mm00445545_m1; Life Technologies), tumour necrosis factor alpha (TNF-alpha) (Mm00443258_m1; Life Technologies), insulin-like growth factor 1 (IGF1) (Mm00439560_m1; Life Technologies), CD4 (Mm00442754_m1; Life Technologies), forkhead box P3 (FOXP3) (Mm00475162_m1; Life Technologies), amphiregulin (Mm00437583_m1; Life Technologies), CD11c (Mm00498701_m1; Life Technologies), interferon-γ (IFNγ) (Mm01168134_m1; Life Technologies), transforming growth factor-β1 (Mm01178820_m1; Life Technologies), CD8a (Mm01182107_g1; Life Technologies), MyoD1 (Mm00440387_m1; Life Technologies), MyoG (Mm00446194_m1; Life Technologies), and Tmem8c (Mm00481256_m1; Life Technologies).
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Publication 2023
Actins Amphiregulin Animals Biological Assay Deoxyribonuclease I DNA, Complementary Gamma Rays Gene Expression Genes Glycoprotein Hormones, alpha Subunit IGF1 protein, human Interferon Type II MYOD1 protein, human Nicotinic Receptors Protein Subunits Real-Time Polymerase Chain Reaction Reverse Transcription TGF-beta1 trizol Tumor Necrosis Factor-alpha
IL-9, EGFR, AREG, and HIF-1α levels were measured by commercial enzyme-linked immunosorbent assay (ELISA) kits purchased from R&D Systems (Minneapolis, MN, USA), including Human IL-9 DuoSet ELISA (Catalog Number: DY209-05), Human EGFR DuoSet ELISA (Catalog Number: DY231), Human Amphiregulin DuoSet ELISA (Catalog Number: DY262), and Human/Mouse Total HIF-1 alpha/HIF1A DuoSet IC ELISA (Catalog Number: DYC1935-2). Briefly, 100 µL of samples or standards were added to the wells of plates and incubated for 2 h at room temperature. The plates were washed five times. One hundred microliters of the detection antibodies were added to each well and incubated for 2 h at room temperature. The plates were washed five times. One hundred microliters of streptavidin-horseradish peroxidases were added to each well and incubated at room temperature for 20 min. The plates were washed five times. One hundred microliters of substrate solutions were added to each well and incubated at room temperature for 20 min. Fifty microliters of stop solutions were added to each well. The optical density of each well was determined using a microplate reader set to 450 nm.
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Publication 2023
Amphiregulin Antibodies AREG protein, human EGFR protein, human Enzyme-Linked Immunosorbent Assay HIF1A protein, human Homo sapiens Horseradish Peroxidase Mus Streptavidin Vision
Oocytes still enclosed in the cumulus cells (CEOs) were injected with 12.5 ng/μl Il7-RL, Thumpd1-RL, or Tpx2-RL and 12.5 ng/µl of polyadenylated FL using a FemtoJet Express programmable microinjector with an Automated Upright Microscope System (Leica, DM4000B). Injected CEOs were pre-incubated for 3 h in culture medium supplemented with 2 μM milrinone, and then cultured in milrinone-free medium supplemented with 100 nM amphiregulin. After 16 h, the CEOs were denuded, collected in lysis buffer and frozen. Luciferase activities in the oocyte extracts were measured using a dual-luciferase reporter assay kit (Promega), and luminescence was detected with a SpectraMax L luminometer (Molecular Devices). Data are reported as ratios of RL and FL.
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Publication 2023
Amphiregulin Biological Assay Buffers Cells Culture Media Cumulus Cells Freezing Luciferases Luminescence Medical Devices Microscopy Milrinone Oocytes Promega TPX2 protein, human
After the treatment period, treated cells or rat lung tissues were subjected to RNA isolation using RNAiso plus as described earlier (Andugulapati et al. 2020 (link)). Briefly, RNA was isolated using the TRIzol–chloroform method and total RNA was quantified using nano-drop. Isolated RNA (1 μg) was subjected to cDNA synthesis using a prime script cDNA synthesis kit as per the manufacturer’s instructions. Specified primers [IL-6, IL-1β, TNF-α, IL-8, CCL2, CCL3, CCL7, CXCL1, alveolar pulmonary stretch (amphiregulin), CC16, vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule (ICAM-1), and reference markers (GAPDH, β2M, and β-actin)] were designed using Primer-3 software and the respective sequences are shown in Table S1. RT-qPCR was carried out using SYBR green mix and the differences in mRNA expression of the specified genes were calculated as the fold change using the formula 2-ΔΔct and data were expressed as mean ± SEM.
Publication 2023
Actins Amphiregulin Anabolism CCL2 protein, human CCL3 protein, human CCL7 protein, human Cells Chloroform CXCL1 protein, human DNA, Complementary GAPDH protein, human Gene Expression Intercellular Adhesion Molecules Interleukin-1 beta Lung Oligonucleotide Primers RNA, Messenger SYBR Green I Tissues trizol Tumor Necrosis Factor-alpha Vascular Cell Adhesion Molecule-1
Cisplatin (cDDP), vitamin E (VitE) and hydralazine (Hlz) were obtained from Sigma (St. Louis, MO, USA) and 4μ8cα was purchased from Millipore (Burlington, MA, USA). MK2206, an AKT inhibitor, was purchased from Selleck (Shanghai, China). Intracellular reactive oxygen species (ROS) were detected via 2′,7′‐dichlorofluorescin diacetate (DCFDA) staining (Thermo Scientific, Waltham, MA, USA). Moreover, 4,4‐difluoro‐1,3,5,7,8‐pentamethyl‐4‐bora‐3a,4a‐diaza‐s‐indacene (BODIPY 493/503) was obtained from Thermo Scientific, and 4‐hydroxy‐trans‐2‐nonenal (4‐HNE)‐protein adducts in the indicated cells were detected through competitive enzyme‐linked immunosorbent assays (ELISAs) (Abcam, Cambridge, UK). Primary antibodies (Abs) against amphiregulin (AREG) (AB‐262‐NA) and basic fibroblast growth factor (bFGF) (AF‐233‐NA) were obtained from R&D Systems (Minneapolis, MN, USA).
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Publication 2023
4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene 4-hydroxy-2-nonenal Amphiregulin Antibodies Cells Cisplatin dichlorofluorescin Enzyme-Linked Immunosorbent Assay Fibroblast Growth Factor 2 Hydralazine MK 2206 Proteins Protoplasm Reactive Oxygen Species Vitamin E

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Amphiregulin is a growth factor that belongs to the epidermal growth factor (EGF) family. It is a soluble protein that can bind to and activate the EGF receptor, promoting cell proliferation and differentiation.
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The RNeasy Mini Kit is a laboratory equipment designed for the purification of total RNA from a variety of sample types, including animal cells, tissues, and other biological materials. The kit utilizes a silica-based membrane technology to selectively bind and isolate RNA molecules, allowing for efficient extraction and recovery of high-quality RNA.
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The Human Amphiregulin Quantikine ELISA Kit is a quantitative sandwich enzyme immunoassay designed for the measurement of human amphiregulin levels in cell culture supernates, serum, and plasma.
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Gefitinib is a tyrosine kinase inhibitor used in laboratory research. It functions by inhibiting the epidermal growth factor receptor (EGFR) tyrosine kinase.
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Diff-Quick is a set of staining solutions used for rapid differential staining of blood smears and other cytological samples. It is a quick and easy-to-use staining method that allows for the identification of different cell types, such as red blood cells, white blood cells, and platelets, in a sample. The Diff-Quick staining procedure involves three steps: fixation, staining, and rinsing. The stained samples can then be examined under a microscope for analysis.

More about "Amphiregulin"

Epidermal Growth Factor, EGF, EGFR, Cell Proliferation, Cell Differentiation, Cell Survival, Tissue Development, Homeostasis, Cancer, Inflammation, DuoSet ELISA, RNeasy Mini Kit, Human Amphiregulin Quantikine ELISA Kit, RNeasy Plus Mini Kit, Gefitinib, PMA, Diff-Quick