ligand for egfr  (R&D Systems)

Journal: PLoS ONE

Article Title: Telmisartan Inhibits Cell Proliferation by Blocking Nuclear Translocation of ProHB-EGF C-Terminal Fragment in Colon Cancer Cells

doi: 10.1371/journal.pone.0056770

Figure Lengend Snippet: Quoted from and modified. TPA induces an ADAM-mediated cleavage of proHB-EGF, and results in the ectodomain shedding of its N-terminal fragment and generation of an intracellular C-terminal fragment (CTF). The soluble HB-EGF binds to the EGFR and induces a rapid transient phosphorylation of EGFR. This phosphorylation results in the transcription of various genes. Meanwhile the HB-EGF-CTF is translocated into the nucleus, where it subsequently induces the nuclear export of PLZF. This results in the progression of cell cycle. The potent inhibitor blocks the nuclear translocation of HB-EGF-CTF. P indicates phosphorylation. Abbreviations: EGFR; epidermal growth factor receptor, TPA; 12- O -tetradecanoylphorbol-13-acetate, PKCδ; protein kinase Cδ, ADAM; a disintegrin and metalloproteinase, HB-EGF; heparin-binding EGF-like growth factor, CTF; C-terminal fragment, MAPK; mitogen-activated protein kinase, PLZF; promyelocytic leukemia zinc finger.

Article Snippet: An Alphascreen system® (PerkinElmer, Shelton, CT, USA) was used to screen a library of 9000 chemical compounds (Carna Bioscience Inc., Kobe, Japan) cyclopaedically for their efficacy in blocking the interaction between biotinylated-EGFR ligands-CTF and GST-Zn5-8 of PLZF.

Techniques: Modification, Translocation Assay, Binding Assay

Journal: PLoS ONE

Article Title: Telmisartan Inhibits Cell Proliferation by Blocking Nuclear Translocation of ProHB-EGF C-Terminal Fragment in Colon Cancer Cells

doi: 10.1371/journal.pone.0056770

Figure Lengend Snippet: (A) Schema of FLAG-tagged full length PLZF consisting of FLAG, BTB, Center, and nine ZnFs. (B) HT1080 cells stably expressing pro-HB-EGF, pro-TGF-α, pro-AR and pro-EPR were transiently transfected with an expression vector encoding FLAG-tagged PLZF. PLZF protein expression of cell lysates (lane1), as well as, cells treated with 100 nM TPA for 1 h, and probed with anti-FLAG antibodies following immunoprecipitation with anti-EGFR ligands-CTF antibodies (lane2) and an anti-normal rabbit IgG (lane3). (C) GST pull-down assay. Cell lysates containing FLAG-tagged PLZF derivatives were incubated with GST (lane 2), GST-HB-EGF-CTF (lane 3), GST-TGF-α-CTF (lane4), GST-AR-CTF (lane 5), and GST-EPR-CTF (lane 6) beads for 2 h, and bound proteins were detected by immunoblotting with an anti-FLAG antibody. (D) Schema of FLAG-tagged PLZF derivatives. The binding properties of PLZF derivatives to GST-fused- HB-EGF-CTF, TGF-α-CTF, AR-CTF and EPR-CTF GST in a pull-down assay, as summarized in the right lanes of each structure. Binding properties are based on the estimation of band intensity with are relative to the control band and indicated by++(>50%),+(50–10%), and − (<10%).

Article Snippet: An Alphascreen system® (PerkinElmer, Shelton, CT, USA) was used to screen a library of 9000 chemical compounds (Carna Bioscience Inc., Kobe, Japan) cyclopaedically for their efficacy in blocking the interaction between biotinylated-EGFR ligands-CTF and GST-Zn5-8 of PLZF.

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Pull Down Assay, Incubation, Western Blot, Binding Assay

Journal: PLoS ONE

Article Title: Telmisartan Inhibits Cell Proliferation by Blocking Nuclear Translocation of ProHB-EGF C-Terminal Fragment in Colon Cancer Cells

doi: 10.1371/journal.pone.0056770

Figure Lengend Snippet: (A) Direct interaction between EGFR-ligand-CTF and PLZF (GST-tagged-ZnF5-8) with the SPR system. The recombinant biotin-EGFR- ligand-CTFs were individually immobilized, by SA sensor chips. GST-Zn5-8 with concentrations ranging from 0.03 to 1.0 µM was then injected with running buffer at 25°C and a flow rate of 30 µL/min for 2 min. (B) The expression vector encoding CFP-PLZF was co-transfected into wild-type HT1080 cells and HT1080 cells stably expressing either proHB-EGF, proTGF-α, proAR or proEPR. The cells were cultured for 24 h and then pretreated in serum-free medium with KB-R7785. Cells were then treated in the serum-free conditioned medium with TPA, and the subcellular localization of the CFP fusion protein was observed. To determine the percentage of cells (mean±SD) demonstrating nuclear localization of CFP-PLZF, cells were counted in at least two transfections, and at least 200 cells expressing CFP-PLZF were examined in each experiment. *P<0.05 for the stimulus effect during TPA treatment vs. no treatment, and **P<0.05 for the inhibitory effect during KB-R7785 treatment vs. TPA treatment. (C) A schematic of the high-throughput Alphascreen system. Upon excitation at 680 nm, ambient oxygen is converted to singlet oxygen ( 1 O 2 ) by a photosensitizer present in the donor beads. If the acceptor beads are in close proximity (<200 nm), 1 O 2 transfers its energy to thioxene derivatives present in the acceptor beads leading to emission of light at 520–620 nm. One complex consists of streptavidin-coated donor beads and biotinylated EGFR ligands-CTF. Another consists of anti-GST antibody-conjugated acceptor beads and GST-ZF5-8. If the association between EGFR ligand-CTF and ZnF5-8 occurs, the beads are close enough to allow detection of a signal. Any inhibitors of this interaction would increase the distance between the beads, and the signal would be lost. (D) Alphascreen signals of GST or GST-Zn5-8 incubated with various concentrations of biotin-HB-EGF-CTF. (E) Alphascreen signals of biotin-HB-EGF-CTF incubated with various concentrations of GST or GST-Zn5-8. (E) The binding abilities of HB-EGF, TGF-α, amphiregulin (AR), and epiregulin (EPR), to PLZF with Alphascreen system.

Article Snippet: An Alphascreen system® (PerkinElmer, Shelton, CT, USA) was used to screen a library of 9000 chemical compounds (Carna Bioscience Inc., Kobe, Japan) cyclopaedically for their efficacy in blocking the interaction between biotinylated-EGFR ligands-CTF and GST-Zn5-8 of PLZF.

Techniques: Recombinant, Injection, Expressing, Plasmid Preparation, Transfection, Stable Transfection, Cell Culture, High Throughput Screening Assay, Amplified Luminescent Proximity Homogenous Assay, Incubation, Binding Assay

Journal: PLoS ONE

Article Title: Telmisartan Inhibits Cell Proliferation by Blocking Nuclear Translocation of ProHB-EGF C-Terminal Fragment in Colon Cancer Cells

doi: 10.1371/journal.pone.0056770

Figure Lengend Snippet: TPA-induced cell proliferation through EGFR and nuclear translocation of HB-EGF-CTF signaling. (A) Growth curve assay. HT29 cell numbers which were counted daily, 24 h (i.e.day1) after cells were seeded in three dependent colonies that were cultured in conditioned media. The values are means of three independent experiments. (B) Cell numbers of colonies cultured in 5% FBS conditioned media with or without TPA, KB-R7785, AG1478, and recombinant HB-EGF on day6. The cells were also observed with microscopy (×200) *P <0.05 for the stimulus effect, and **P<0.05 for the inhibitory effect. (C) Effects of KB-R7785 and AG1478 on TPA-induced nuclear translocation of HB-EGF-CTF and nuclear export of PLZF. Cells were treated with TPA following preincubation with or without KB-R7785 and AG1478. Immunofluorescent staining with anti-HB-EGF-CTF antibodies (red signals), anti-PLZF antibodies (green) and DAPI (blue), which stains for nuclei was performed. Images were obtained on a fluorescence microscope (×200). The white bar indicated 10 µm. (D) Effects of KB-R7785 on the association between HB-EGF-CTF and PLZF after TPA stimulation. Cells were treated with TPA at various times following preincubation with or without KB-R7785. Blotted samples were probed with antibodies against PLZF after immunoprecipitation with anti-HB-EGF-CTF antibody ( upper panel ). The total amount of HB-EGF-CTF in the immunoprecipitates was determined by reprobing the same blot with an anti-HB-EGF antibody ( lower panel ).

Article Snippet: An Alphascreen system® (PerkinElmer, Shelton, CT, USA) was used to screen a library of 9000 chemical compounds (Carna Bioscience Inc., Kobe, Japan) cyclopaedically for their efficacy in blocking the interaction between biotinylated-EGFR ligands-CTF and GST-Zn5-8 of PLZF.

Techniques: Translocation Assay, Cell Culture, Recombinant, Microscopy, Staining, Fluorescence, Immunoprecipitation

Journal: PLoS ONE

Article Title: Telmisartan Inhibits Cell Proliferation by Blocking Nuclear Translocation of ProHB-EGF C-Terminal Fragment in Colon Cancer Cells

doi: 10.1371/journal.pone.0056770

Figure Lengend Snippet: (A) Effects of telmisartan or candesartan on TPA-induced EGFR phosphorylation. Cells were preincubated with or without telmisartan or candesartan, and then treated with TPA for 0, 15, 60 and 120 min. Blotted samples were probed with an anti-phosphotyrosine antibody after immunoprecipitation with an anti-EGFR antibody ( upper panel ). The total amount of EGFR in the immunoprecipitates was determined by reprobing the same blot with an anti-EGFR antibody ( lower panel ). (B) Effects of telmisartan or candesartan on TPA-induced nuclear translocation of HB-EGF-CTF and nuclear export of PLZF following knockdown of AT1R with siRNA. Probing with an anti-AT1R antibody ( upper panel ) and anti-β-actin antibody (lower panel ). (C) Cells were then treated with TPA following preincubation with or without telmisartan or candesartan. Immunofluorescent stainings with anti-HB-EGF-CTF antibodies (red), anti-PLZF antibodies (green) and DAPI (blue) were performed following knockdown of AT1R with siRNA. Images were obtained on a fluorescence microscope (×400). The white bar indicated 10 µm. (D)The inhibitory effects of telmisartan and candesartan on TPA-induced cell proliferation in HT29 cells with CCK-8 kit assay following knockdown of AT1R with siRNA. *P<0.05 for the stimulus effect, and **P<0.05 for the inhibitory effect.

Article Snippet: An Alphascreen system® (PerkinElmer, Shelton, CT, USA) was used to screen a library of 9000 chemical compounds (Carna Bioscience Inc., Kobe, Japan) cyclopaedically for their efficacy in blocking the interaction between biotinylated-EGFR ligands-CTF and GST-Zn5-8 of PLZF.

Techniques: Immunoprecipitation, Translocation Assay, Fluorescence, Microscopy, CCK-8 Assay

Journal: PLOS One

Article Title: EGFR-targeted affibody–polyIC polyplex kills EGFR-overexpressing cancer cells without activating the EGFR

doi: 10.1371/journal.pone.0334584

Figure Lengend Snippet: (A i) SDS-PAGE analysis showing the purity of the Ni-NTA purified Z EGFR 1907’ under pseudo native and denatured conditions. Pseudo native gel bands (boxed) were extracted and analysed by mass spectrometry. Mass spectrometry data confirmed that both bands correspond to one species, (A ii) DNA sequencing of the plasmid revealed the above sequence and this was also confirmed by mass spectrometry analysis (B i) Elution profile of the size exclusion gel filtration chromatography of Z EGFR 1907’ affibody purified via His-Tag Ni-NTA purification. Superdex 75 1660 column equilibrated with 20 mM Hepes pH 7.4, 500 mM NaCl, 10% Glycerol and 2 mM β mercaptoethanol and 1 ml fractions were collected. Insert shows the complete elution profile. (B ii) 20 µl of selected sample fractions from the purification were denatured, run on an SDS-PAGE gel and stained with Coomassie blue. (C) Silver stained SDS-PAGE gel of the concentrated 1:1 triconjugate Z EGFR 1907’ (PPEA) under pseudo native and denatured conditions. Pseudo native and denatured conditions are indicated by -/+ β-mercaptoethanol.

Article Snippet: Many common cancers over-express the EGFR, by attaching polyIC to PEG and the poly-imine there is less non-specific cellular uptake; the addition of the high affinity EGFR ligand (i.e., the affibody) then directs the polyIC to the tumor cells.

Techniques: SDS Page, Purification, Mass Spectrometry, DNA Sequencing, Plasmid Preparation, Sequencing, Filtration, Chromatography, Staining

Journal: PLOS One

Article Title: EGFR-targeted affibody–polyIC polyplex kills EGFR-overexpressing cancer cells without activating the EGFR

doi: 10.1371/journal.pone.0334584

Figure Lengend Snippet: (A) PPEA-polyplexes selectively kill cell lines overexpressing EGFR. Cells were seeded in duplicates into 96-well plates at a density of 5000 cells in 0.1 ml medium per well and grown overnight. Cells were then treated with polyIC at the indicated concentrations using the PPEA complex. PEI-PEG ratio = 1:1; w/w ratio PEI: polyIC = 0.78. U138MG cells do not express EGFR; U87MGwtEGFR cells express 1x10 6 , A431 express 2-3x10 6 and MDA-MB-468 express 2x10 6 EGFRs/cell. (B,C) In vitro anti-tumor activity of PPEA-polyIC-polyplex is polyIC-specific. A431 and U87MGwtEGFR cell lines were treated with same doses of PPEA-polyIC-polyplex (B) or PPEA polyI polyplex (C) , which served as negative control. Viability was measured by the PrestoBlue Cell Viability Reagent (Invitrogen), according to the manufacturer’s instructions, at 72 hrs after treatment. These experiments were repeated three times with a representative experiment shown.

Article Snippet: Many common cancers over-express the EGFR, by attaching polyIC to PEG and the poly-imine there is less non-specific cellular uptake; the addition of the high affinity EGFR ligand (i.e., the affibody) then directs the polyIC to the tumor cells.

Techniques: In Vitro, Activity Assay, Negative Control

Journal: bioRxiv

Article Title: Extracellular signalling regulates gastrin transcription through site-specific phosphorylation and nuclear redistribution of Menin

doi: 10.64898/2026.04.07.717082

Figure Lengend Snippet: (A) Hematoxylin and eosin (H&E) staining of normal duodenum containing Brunner’s glands (nDUO-BG) and duodenal neuroendocrine tumor (DNET). Dashed boxes indicate regions shown at higher magnification. (B) Immunohistochemical staining for synaptophysin (SYP) confirming neuroendocrine differentiation in DNET. (C-D) Immunohistochemical staining for TGFα and EREG in tumor-associated Brunner’s glands (tBG) and DNET. Dashed boxes indicate tumor-gland interfaces. (E-F) Quantification of TGFα and EREG expression by H-score in nDUO-BG, tBG, and DNET. Data are mean ± SEM; ns, not significant; ****P < 0.0001. (G) EGFR immunostaining in nDUO-BG and DNET showing heterogeneous expression across tissues. (H) Menin immunostaining in nDUO-BG and DNET. (I) Representative FFPE DNET specimens showing cytoplasmic or near-absent Menin expression, accompanied by strong TGFα and EREG staining within tumor cells. (J) Quantification of Menin nuclear-to-cytoplasmic (N/C) ratio in nDUO-BG and DNET. Data are mean ± SEM; ****P < 0.0001. Images were taken at 100X, 200X and 400X. Scale bars: 100 μm (low magnification) and 50 μm (high magnification).

Article Snippet: After 24 h, cells were serum-starved for 24 h, followed by treatment with either recombinant human eregulin (EREG) protein (10 nM), a potent ligand for EGFR (R&D systems, #1195-EP) or 10 μM Forskolin (FSK, ThermoFisher, #66575-29-9), activator of adenylyl cyclase and cyclic AMP or and 10nM phorbol 12-myristate 13-acetate (TPA; Sigma-Aldrich, #P8139) for 4-8 h. Cells were lysed, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, #E1980) according to the manufacturer’s instructions.

Techniques: Staining, Immunohistochemical staining, Expressing, Immunostaining

Journal: bioRxiv

Article Title: Extracellular signalling regulates gastrin transcription through site-specific phosphorylation and nuclear redistribution of Menin

doi: 10.64898/2026.04.07.717082

Figure Lengend Snippet: (A) Multiple sequence alignment of the Menin C-terminal region from the indicated vertebrate species showing strong conservation of a basic residue–rich motif encompassing Ser487. Conserved basic residues and Ser487 are highlighted. (B) Schematic of human Menin illustrating the position of Ser487 within NLS1. The expanded sequence highlights Ser487 and surrounding basic residues; constructs used in this study. (C) Immunoblot analysis of AGS cells expressing FLAG-tagged wild-type Menin or Ser487 mutants (S487A, S487D) following treatment with EREG, FSK, or TPA. Whole-cell lysates were probed with antibodies against phospho-Ser487 Menin, FLAG-Menin, and GAPDH. (D, E) Immunoblot analysis of MKN-45G and KATO III cells expressing wild-type Menin following stimulation with EREG, FSK, or TPA. Blots were probed for phospho-Ser487 Menin, FLAG-Menin, and β-tubulin. (F, H) Quantification of phospho-Ser487 Menin in AGS, KATO III and MKN-45G cells. (I) Immunoblot analysis of AGS cells examining activation of cAMP and EGFR downstream kinases under the indicated conditions. (J) Densitometric quantification of signalling outputs shown in (I), expressed as fold change relative to vector control. (K) Time-course of Ser487 phosphorylation in AGS cells stimulated with TPA in the presence of kinase inhibitors; MEK inhibitor (U0126), AKT inhibitor (MK-2206), PKC inhibitor (Gö6983), or combined MEK+AKT inhibition. (L) Quantification of Ser487 phosphorylation kinetics following TPA stimulation with the indicated inhibitors. (M) Area-under-the-curve (AUC) analysis of phosphorylation in (L). Data are presented as mean ± SEM; individual data points represent independent biological replicates (n = 3). Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparison test (*P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant).

Article Snippet: After 24 h, cells were serum-starved for 24 h, followed by treatment with either recombinant human eregulin (EREG) protein (10 nM), a potent ligand for EGFR (R&D systems, #1195-EP) or 10 μM Forskolin (FSK, ThermoFisher, #66575-29-9), activator of adenylyl cyclase and cyclic AMP or and 10nM phorbol 12-myristate 13-acetate (TPA; Sigma-Aldrich, #P8139) for 4-8 h. Cells were lysed, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, #E1980) according to the manufacturer’s instructions.

Techniques: Sequencing, Residue, Construct, Western Blot, Expressing, Activation Assay, Plasmid Preparation, Control, Phospho-proteomics, Inhibition, Comparison

Journal: Protein Science : A Publication of the Protein Society

Article Title: Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics

doi: 10.1002/pro.5141

Figure Lengend Snippet: (Left) Depiction of EGFR in the inactive monomeric state adopting the tethered conformation prior to EGF induced dimerization of EGFR with another EGF bound receptor. The ectodomain of EGFR includes the ligand binding domain of EGFR while the tyrosine kinase domain is intracellular. Created with biorender.com. (Right) Cartoon depiction of active EGFR domains I–IV, and sites 1–3.

Article Snippet: The three sidechain–sidechain interactions conserved among other endogenous EGFR ligands have been experimentally characterized as critical for EGF–EGFR binding (Engler et al., ; Matsunami et al., ; Tadaki & Niyogi, ).

Techniques: Ligand Binding Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics

doi: 10.1002/pro.5141

Figure Lengend Snippet: Monomeric EGFR exits in the tethered conformation. When EGF binds EGFR, EGFR undergoes conformational changes enabling the formation of an active EGFR dimer. Antibodies such as cetuximab and matuzumab bind tethered EGFR and prevent EGFR dimerization. This figure was created with PDB files 1YY9, 3C09, and 1IVO.

Article Snippet: The three sidechain–sidechain interactions conserved among other endogenous EGFR ligands have been experimentally characterized as critical for EGF–EGFR binding (Engler et al., ; Matsunami et al., ; Tadaki & Niyogi, ).

Techniques:

Journal: Protein Science : A Publication of the Protein Society

Article Title: Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics

doi: 10.1002/pro.5141

Figure Lengend Snippet: Plots of EGFR single residue alanine scan change in K D data against the Rosetta interaction energy for the given residue in the binding complex. (a) Includes all single alanine mutations for duligotuzumab (Schaefer et al., ), matuzumab (Schmiedel et al., ), cetuximab and necitumumab (Li et al., ). (b) Excludes lysine residues from the analysis.

Article Snippet: The three sidechain–sidechain interactions conserved among other endogenous EGFR ligands have been experimentally characterized as critical for EGF–EGFR binding (Engler et al., ; Matsunami et al., ; Tadaki & Niyogi, ).

Techniques: Residue, Binding Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics

doi: 10.1002/pro.5141

Figure Lengend Snippet: The crystal structure of EGF bound to EGFR (PDB ID: 1IVO). Residues that make important binding interactions are shown. Dashed lines indicate hydrogen bonding and salt bridge interactions.

Article Snippet: The three sidechain–sidechain interactions conserved among other endogenous EGFR ligands have been experimentally characterized as critical for EGF–EGFR binding (Engler et al., ; Matsunami et al., ; Tadaki & Niyogi, ).

Techniques: Binding Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics

doi: 10.1002/pro.5141

Figure Lengend Snippet: Conserved interactions between EGFR and EGF (PDB 1IVO), TGF‐α (PDB 1MOX), Epigen (PDB 5WB8), and Epiregulin (PDB 5WB7). Site 3 interactions left. Site 2 interactions right. Residues that make important binding interactions are shown. Dashed lines indicate hydrogen bonding and salt bridge interactions.

Article Snippet: The three sidechain–sidechain interactions conserved among other endogenous EGFR ligands have been experimentally characterized as critical for EGF–EGFR binding (Engler et al., ; Matsunami et al., ; Tadaki & Niyogi, ).

Techniques: Binding Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics

doi: 10.1002/pro.5141

Figure Lengend Snippet: The Rosetta energy breakdown of the energy contribution between EGF, TGF‐ α, epigen, and epiregulin with the EGFR residues that they interact with.

Article Snippet: The three sidechain–sidechain interactions conserved among other endogenous EGFR ligands have been experimentally characterized as critical for EGF–EGFR binding (Engler et al., ; Matsunami et al., ; Tadaki & Niyogi, ).

Techniques: Residue

Journal: Protein Science : A Publication of the Protein Society

Article Title: Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics

doi: 10.1002/pro.5141

Figure Lengend Snippet: EGF and TGF‐α differ in their ability to stabilize the tips separated conformation of EGFR as illustrated by cryo EM studies (PDB 7SYD, 7SYE, 7SZ5, and 7SZ7). While EGF stabilizes a symmetric dimer (PDB 3NJP) dimer with EGFR, Epiregulin (PDB 5WB7) stabilized an asymmetric dimer with bent and unbent EGFR conformations, and Epigen (PDB 5WB8) forms very weak dimer with EGFR and crystallizes as a monomer with EGFR. For EGFR multimers, EGFR domain IV is the proposed multimerization interface (Huang et al., ).

Article Snippet: The three sidechain–sidechain interactions conserved among other endogenous EGFR ligands have been experimentally characterized as critical for EGF–EGFR binding (Engler et al., ; Matsunami et al., ; Tadaki & Niyogi, ).

Techniques: Cryo-EM Sample Prep

Journal: Protein Science : A Publication of the Protein Society

Article Title: Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics

doi: 10.1002/pro.5141

Figure Lengend Snippet: Domain I of EGFR colored by the per‐residue interaction energy with a variety of binders. Energy units are Rosetta Energy Units (REU). White represents no interaction. Red represents an unfavorable interaction. Blue represents a favorable interaction.

Article Snippet: The three sidechain–sidechain interactions conserved among other endogenous EGFR ligands have been experimentally characterized as critical for EGF–EGFR binding (Engler et al., ; Matsunami et al., ; Tadaki & Niyogi, ).

Techniques: Residue

Journal: Protein Science : A Publication of the Protein Society

Article Title: Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics

doi: 10.1002/pro.5141

Figure Lengend Snippet: The Rosetta energy breakdown of the energy contribution between EGF, TGF‐α, epigen, epiregulin, and adnectin 1 (ADN1) with the EGFR residues that they interact with.

Article Snippet: The three sidechain–sidechain interactions conserved among other endogenous EGFR ligands have been experimentally characterized as critical for EGF–EGFR binding (Engler et al., ; Matsunami et al., ; Tadaki & Niyogi, ).

Techniques: Residue

Journal: Protein Science : A Publication of the Protein Society

Article Title: Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics

doi: 10.1002/pro.5141

Figure Lengend Snippet: Interactions between EGFR domain I and EGF (PDB 1IVO), TGF‐α (PDB 1MOX), Epigen (PDB 5WB8), Epiregulin (PDB 5WB7), and Adnectin 1 (PDB 3QWQ). Residues that make important binding interactions are shown. Dashed lines indicate hydrogen bonding and salt bridge interactions.

Article Snippet: The three sidechain–sidechain interactions conserved among other endogenous EGFR ligands have been experimentally characterized as critical for EGF–EGFR binding (Engler et al., ; Matsunami et al., ; Tadaki & Niyogi, ).

Techniques: Binding Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics

doi: 10.1002/pro.5141

Figure Lengend Snippet: Affinity, interaction surface area, and shape complementary for EGFR binders.

Article Snippet: The three sidechain–sidechain interactions conserved among other endogenous EGFR ligands have been experimentally characterized as critical for EGF–EGFR binding (Engler et al., ; Matsunami et al., ; Tadaki & Niyogi, ).

Techniques:

Journal: Protein Science : A Publication of the Protein Society

Article Title: Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics

doi: 10.1002/pro.5141

Figure Lengend Snippet: Domain III of EGFR colored by the per‐residue interaction energy with a variety of binders. Energy units are Rosetta Energy Units (REU). White represents no interaction. Red represents an unfavorable interaction. Blue represents a favorable interaction.

Article Snippet: The three sidechain–sidechain interactions conserved among other endogenous EGFR ligands have been experimentally characterized as critical for EGF–EGFR binding (Engler et al., ; Matsunami et al., ; Tadaki & Niyogi, ).

Techniques: Residue

Journal: Protein Science : A Publication of the Protein Society

Article Title: Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics

doi: 10.1002/pro.5141

Figure Lengend Snippet: The Rosetta energy breakdown of the energy contribution between EGF, GC1118, 7D12, and 059‐152 with the EGFR residues that they interact with.

Article Snippet: The three sidechain–sidechain interactions conserved among other endogenous EGFR ligands have been experimentally characterized as critical for EGF–EGFR binding (Engler et al., ; Matsunami et al., ; Tadaki & Niyogi, ).

Techniques: Residue

Journal: Protein Science : A Publication of the Protein Society

Article Title: Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics

doi: 10.1002/pro.5141

Figure Lengend Snippet: The EGFR–GC1118 (PDB 4UV7), 7D12 (PDB 4KRL), and 059‐152 (PDB 5XWD) interfaces. Residues that make important binding interactions are shown. Dashed lines indicate hydrogen bonding interactions.

Article Snippet: The three sidechain–sidechain interactions conserved among other endogenous EGFR ligands have been experimentally characterized as critical for EGF–EGFR binding (Engler et al., ; Matsunami et al., ; Tadaki & Niyogi, ).

Techniques: Binding Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics

doi: 10.1002/pro.5141

Figure Lengend Snippet: The EGFR–cetuximab (PDB 1YY9), pantimumab (PDB 5SX4), necitumumab (PDB 3B2U), duligotuzumab (PDB 3P0Y), relaxed 059‐152 (PDB 5XWD), and repebody (PDB 4UIP) interfaces separated into overview, site 3 central and site 3 edge interactions. Residues that make important binding interactions are shown. Dashed lines indicate hydrogen bonding interactions.

Article Snippet: The three sidechain–sidechain interactions conserved among other endogenous EGFR ligands have been experimentally characterized as critical for EGF–EGFR binding (Engler et al., ; Matsunami et al., ; Tadaki & Niyogi, ).

Techniques: Binding Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics

doi: 10.1002/pro.5141

Figure Lengend Snippet: The Rosetta energy breakdown of the energy contribution between EGF, cetuximab (CET), duligotuzumab (DUL), necitumumab (NEC), panitumumab (PAN), 059‐152, and the repebody (REP) with the EGFR residues that they interact with.

Article Snippet: The three sidechain–sidechain interactions conserved among other endogenous EGFR ligands have been experimentally characterized as critical for EGF–EGFR binding (Engler et al., ; Matsunami et al., ; Tadaki & Niyogi, ).

Techniques: Residue

Journal: Protein Science : A Publication of the Protein Society

Article Title: Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics

doi: 10.1002/pro.5141

Figure Lengend Snippet: Domain III of EGFR colored by the per‐residue interaction energy with a variety of binders. Energy units are Rosetta Energy Units (REU). White represents no interaction. Red represents an unfavorable interaction. Blue represents a favorable interaction.

Article Snippet: The three sidechain–sidechain interactions conserved among other endogenous EGFR ligands have been experimentally characterized as critical for EGF–EGFR binding (Engler et al., ; Matsunami et al., ; Tadaki & Niyogi, ).

Techniques: Residue

Journal: Protein Science : A Publication of the Protein Society

Article Title: Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics

doi: 10.1002/pro.5141

Figure Lengend Snippet: The Rosetta energy breakdown of the energy contribution between matuzumab (MAT), EgA1, and 9G8 with the EGFR residues that they interact with.

Article Snippet: The three sidechain–sidechain interactions conserved among other endogenous EGFR ligands have been experimentally characterized as critical for EGF–EGFR binding (Engler et al., ; Matsunami et al., ; Tadaki & Niyogi, ).

Techniques: Residue

Journal: Protein Science : A Publication of the Protein Society

Article Title: Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics

doi: 10.1002/pro.5141

Figure Lengend Snippet: The relaxed EGFR (blue) and matuzumab (pink) interface (PDB 3C09). Residues that make important binding interactions are shown. Dashed lines indicate hydrogen bonding interactions.

Article Snippet: The three sidechain–sidechain interactions conserved among other endogenous EGFR ligands have been experimentally characterized as critical for EGF–EGFR binding (Engler et al., ; Matsunami et al., ; Tadaki & Niyogi, ).

Techniques: Binding Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Analysis of EGFR binding hotspots for design of new EGFR inhibitory biologics

doi: 10.1002/pro.5141

Figure Lengend Snippet: The EGFR–9G8 (PDB 4KRP) and relaxed EgA1(PDB 4KRO) interfaces. Residues that make important binding interactions are shown. Dashed lines indicate hydrogen bonding interactions.

Article Snippet: The three sidechain–sidechain interactions conserved among other endogenous EGFR ligands have been experimentally characterized as critical for EGF–EGFR binding (Engler et al., ; Matsunami et al., ; Tadaki & Niyogi, ).

Techniques: Binding Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Proximal Tubule–Derived Amphiregulin Amplifies and Integrates Profibrotic EGF Receptor Signals in Kidney Fibrosis

doi: 10.1681/ASN.2019030321

Figure Lengend Snippet: Low-affinity EGFR ligands induce sustained EGFR phosphorylation in HPTCs. Equimolar amounts (17 pM) of each ligand were used to stimulate HPTCs for the time points indicated. EGFR phosphorylation was examined by (A) Western blot and (B) densitometric analysis was performed after normalization to nonstimulated cells. Tubulin was used for normalization (loading control). Western blots and quantifications shown are representative of three independent experiments.

Article Snippet: Equimolar (17 pM) or increasing (85 fM–170 pM) amounts of different soluble EGFR ligands (all from R&D Systems) were added to the cells for different time points as noted.

Techniques: Western Blot

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Proximal Tubule–Derived Amphiregulin Amplifies and Integrates Profibrotic EGF Receptor Signals in Kidney Fibrosis

doi: 10.1681/ASN.2019030321

Figure Lengend Snippet: Sustained EGFR reactivation requires transcription and release of endogenous AREG. (A and B) HPTCs were pretreated with DMSO or BB94 (10 ∝M) for 30 minutes and then treated with different low-affinity EGFR ligands for 24 hours. (A) EGFR phosphorylation and downstream ERK1/2 phosphorylation was quantified by Western blot (left panel) followed by densitometric analysis (graph) after normalization to control-stimulated cells (% Control). (B) Endogenous sAREG released in cell culture medium was measured by ELISA and presented as percentile of sAREG concentration in control-stimulated cell medium (% Control). (C) HPTCs were treated with different EGFR ligands (shown in the legend) for 24 hours and mRNA expression of endogenous EGFR ligands (shown in x-axis) was tested by quantitative PCR. Results are presented after normalization to non-stimulated cells (fold control). (D) HPTCs were treated with different EGFR ligands for 4 hours and then culture medium was changed to fresh medium containing AREG-neutralization antibody or vehicle for 20 hours. EGFR phosphorylation was quantified by Western blot (left panel) and by densitometric analysis (right column graph) after normalization to control-stimulated/vehicle-treated cells (% Control). Tubulin was used as loading control in (A and D). For all experiments, n=3–6. *P<0.05; **P<0.01.

Article Snippet: Equimolar (17 pM) or increasing (85 fM–170 pM) amounts of different soluble EGFR ligands (all from R&D Systems) were added to the cells for different time points as noted.

Techniques: Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay, Expressing, Real-time Polymerase Chain Reaction, Neutralization

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Proximal Tubule–Derived Amphiregulin Amplifies and Integrates Profibrotic EGF Receptor Signals in Kidney Fibrosis

doi: 10.1681/ASN.2019030321

Figure Lengend Snippet: AREG transcriptional upregulation and sustained EGFR reactivation require YAP1. (A) Cells were control-treated or treated with sAREG for 24 hours and total YAP1 or phospho-YAP1 (pYAP1) levels were examined by Western blot in whole cell lysates (left panel). Quantification of phospho-YAP1 and total YAP1 levels, as well as the ratio of phospho-to-total YAP1, is presented in the graphs after densitometric analysis (right panels). Tubulin was used as loading control. (B) HPTCs were control-treated or treated with sAREG for 24 hours and preparations of nuclear and cytoplasmic fractions were analyzed by Western blot (left panels). Tubulin was used as loading control for cytoplasmic fractions and histone 2A was used as loading control for nuclear fractions. Quantification of total YAP1 levels is presented after densitometric analysis (right panel graph). (C and D) HPTCs were transfected with control siRNA (siControl) or siRNA against YAP1 (siYAP1) and at 48 hours post-transfection, serum-starved cells were control-treated or treated with sAREG for 24 hours. Endogenous AREG and YAP1 expression was tested by quantitative PCR ([C], results presented as fold of siControl/control-treated cells) and release of endogenous sAREG to the cell culture medium was tested by ELISA ([D], presented as percent siControl/control-treated cells). Results are presented after normalization for siControl transfected/control-stimulated cells. (E) Control or siYAP1 transfected cells were treated at 48 hours post-transfection with different EGFR ligands for 24 hours and EGFR phosphorylation was tested by Western blot (left panel). Densitometric analysis is presented after normalization for siControl transfected/control-stimulated cells (right panel graph, stars denote significant difference from the respective ligand-treated siControl sample). (F and G) ADAM17 PTC-KO mice or their ADAM17 WT littermates were subjected to IRI or sham surgery and after 5 days their kidneys were collected and tdTomato+ PTCs were sorted by FACS for subsequent mRNA extraction and quantitative PCR analysis of (F) AREG and (G) YAP1 expression. Results are presented after normalization to PTCs from ADAM17 WT/sham surgery mice (% Control). n=3–4; *P<0.05, **P<0.01.

Article Snippet: Equimolar (17 pM) or increasing (85 fM–170 pM) amounts of different soluble EGFR ligands (all from R&D Systems) were added to the cells for different time points as noted.

Techniques: Western Blot, Transfection, Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay, Extraction