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  • 99
    Name:
    EGF Receptor Antibody
    Description:
    The epidermal growth factor EGF receptor is a transmembrane tyrosine kinase that belongs to the HER ErbB protein family Ligand binding results in receptor dimerization autophosphorylation activation of downstream signaling internalization and lysosomal degradation 1 2 Phosphorylation of EGF receptor EGFR at Tyr845 in the kinase domain is implicated in stabilizing the activation loop maintaining the active state enzyme and providing a binding surface for substrate proteins 3 4 c Src is involved in phosphorylation of EGFR at Tyr845 5 The SH2 domain of PLCγ binds at phospho Tyr992 resulting in activation of PLCγ mediated downstream signaling 6 Phosphorylation of EGFR at Tyr1045 creates a major docking site for the adaptor protein c Cbl leading to receptor ubiquitination and degradation following EGFR activation 7 8 The GRB2 adaptor protein binds activated EGFR at phospho Tyr1068 9 A pair of phosphorylated EGFR residues Tyr1148 and Tyr1173 provide a docking site for the Shc scaffold protein with both sites involved in MAP kinase signaling activation 2 Phosphorylation of EGFR at specific serine and threonine residues attenuates EGFR kinase activity EGFR carboxy terminal residues Ser1046 and Ser1047 are phosphorylated by CaM kinase II mutation of either of these serines results in upregulated EGFR tyrosine autophosphorylation 10
    Catalog Number:
    2232
    Price:
    None
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Tyr1068 of human EGF receptor. Antibodies are purified by protein A and peptide affinity chromatography.
    Reactivity:
    Human Mouse Rat Monkey
    Applications:
    Western Blot, Immunoprecipitation
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc anti egfr
    <t>EGFR</t> and <t>ERK</t> phosphorylation. (A) Addition of 1 nM ET-1 to NCI-H838 cells for 2 min moderately increased EGFR and ERK tyrosine phosphorylation, whereas 10, 100 or 1000 nM ET-1 strongly increased EGFR and ERK phosphorylation. (B) The mean % EGFR (□) and ERK (■) tyrosine phosphorylation ± S.D. is indicated for 3 determinations as a function of ET-1 concentration; p
    The epidermal growth factor EGF receptor is a transmembrane tyrosine kinase that belongs to the HER ErbB protein family Ligand binding results in receptor dimerization autophosphorylation activation of downstream signaling internalization and lysosomal degradation 1 2 Phosphorylation of EGF receptor EGFR at Tyr845 in the kinase domain is implicated in stabilizing the activation loop maintaining the active state enzyme and providing a binding surface for substrate proteins 3 4 c Src is involved in phosphorylation of EGFR at Tyr845 5 The SH2 domain of PLCγ binds at phospho Tyr992 resulting in activation of PLCγ mediated downstream signaling 6 Phosphorylation of EGFR at Tyr1045 creates a major docking site for the adaptor protein c Cbl leading to receptor ubiquitination and degradation following EGFR activation 7 8 The GRB2 adaptor protein binds activated EGFR at phospho Tyr1068 9 A pair of phosphorylated EGFR residues Tyr1148 and Tyr1173 provide a docking site for the Shc scaffold protein with both sites involved in MAP kinase signaling activation 2 Phosphorylation of EGFR at specific serine and threonine residues attenuates EGFR kinase activity EGFR carboxy terminal residues Ser1046 and Ser1047 are phosphorylated by CaM kinase II mutation of either of these serines results in upregulated EGFR tyrosine autophosphorylation 10
    https://www.bioz.com/result/anti egfr/product/Cell Signaling Technology Inc
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti egfr - by Bioz Stars, 2021-05
    99/100 stars

    Images

    1) Product Images from "Endothelin causes transactivation of the EGFR and HER2 in non-small cell lung cancer cells"

    Article Title: Endothelin causes transactivation of the EGFR and HER2 in non-small cell lung cancer cells

    Journal: Peptides

    doi: 10.1016/j.peptides.2017.01.012

    EGFR and ERK phosphorylation. (A) Addition of 1 nM ET-1 to NCI-H838 cells for 2 min moderately increased EGFR and ERK tyrosine phosphorylation, whereas 10, 100 or 1000 nM ET-1 strongly increased EGFR and ERK phosphorylation. (B) The mean % EGFR (□) and ERK (■) tyrosine phosphorylation ± S.D. is indicated for 3 determinations as a function of ET-1 concentration; p
    Figure Legend Snippet: EGFR and ERK phosphorylation. (A) Addition of 1 nM ET-1 to NCI-H838 cells for 2 min moderately increased EGFR and ERK tyrosine phosphorylation, whereas 10, 100 or 1000 nM ET-1 strongly increased EGFR and ERK phosphorylation. (B) The mean % EGFR (□) and ERK (■) tyrosine phosphorylation ± S.D. is indicated for 3 determinations as a function of ET-1 concentration; p

    Techniques Used: Concentration Assay

    2) Product Images from "Salmonella Infection Upregulates the Leaky Protein Claudin-2 in Intestinal Epithelial Cells"

    Article Title: Salmonella Infection Upregulates the Leaky Protein Claudin-2 in Intestinal Epithelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0058606

    Salmonella -induced claudin-2 expression is dependent on the SAPK/JNK pathway. Claudin-2 protein expression induced by pathogenic Salmonella in SKCO15 cells was tested by different treatments in (A), (B) and (C). (A) Treatment with the EGFR inhibitor (Gefitinib, 50 ng/ml) for 1 hour, followed by colonization of Salmonella for 30 minutes and incubation for 4 hours. (B) Treatment with the SAPK/JNK inhibitor (SP600125, 50 µM) for 1 hour, followed by colonization of Salmonella for 30 minutes and incubation for 4 hours. (C) Treatment with the PI3K inhibitor (Wortmannin, 50 ng/ml) for 1 hour, followed by colonization of Salmonella for 30 min and incubation for 4 hours. (D) The number of invasive bacteria in the intestinal epithelial cells with the SAPK/JNK (SP600125) and the EGFR (Gefitinib) inhibitors. The SAPK/JNK and EGFR inhibitors can decrease the number of invasive bacteria. Data are expressed as the mean ± SD. *P
    Figure Legend Snippet: Salmonella -induced claudin-2 expression is dependent on the SAPK/JNK pathway. Claudin-2 protein expression induced by pathogenic Salmonella in SKCO15 cells was tested by different treatments in (A), (B) and (C). (A) Treatment with the EGFR inhibitor (Gefitinib, 50 ng/ml) for 1 hour, followed by colonization of Salmonella for 30 minutes and incubation for 4 hours. (B) Treatment with the SAPK/JNK inhibitor (SP600125, 50 µM) for 1 hour, followed by colonization of Salmonella for 30 minutes and incubation for 4 hours. (C) Treatment with the PI3K inhibitor (Wortmannin, 50 ng/ml) for 1 hour, followed by colonization of Salmonella for 30 min and incubation for 4 hours. (D) The number of invasive bacteria in the intestinal epithelial cells with the SAPK/JNK (SP600125) and the EGFR (Gefitinib) inhibitors. The SAPK/JNK and EGFR inhibitors can decrease the number of invasive bacteria. Data are expressed as the mean ± SD. *P

    Techniques Used: Expressing, Incubation

    3) Product Images from "Expression of Voltage-Gated Sodium Channel Nav1.8 in Human Prostate Cancer is Associated with High Histological Grade"

    Article Title: Expression of Voltage-Gated Sodium Channel Nav1.8 in Human Prostate Cancer is Associated with High Histological Grade

    Journal: Journal of clinical & experimental oncology

    doi: 10.4172/2324-9110.1000102

    Na v 1.8 is localized to the nucleus Human prostate cancer cell lines were fractionated into plasma membrane, cytosolic and nuclear enriched fractions. Proteins were separated on a 4-12% SDS-PAGE gradient gel. A. Subcellular fractions of PC-3 cells immunoblotted with Na v 1.8. B. Subcellular fractions of C4-2 cells immunoblotting with Na v 1.1 and 1.7 and 1.8. Purity of the enriched fractions were shown using anti-α-tubulin for the cytoplasm, EGFR for the plasma membrane, and PARP for the nucleus.
    Figure Legend Snippet: Na v 1.8 is localized to the nucleus Human prostate cancer cell lines were fractionated into plasma membrane, cytosolic and nuclear enriched fractions. Proteins were separated on a 4-12% SDS-PAGE gradient gel. A. Subcellular fractions of PC-3 cells immunoblotted with Na v 1.8. B. Subcellular fractions of C4-2 cells immunoblotting with Na v 1.1 and 1.7 and 1.8. Purity of the enriched fractions were shown using anti-α-tubulin for the cytoplasm, EGFR for the plasma membrane, and PARP for the nucleus.

    Techniques Used: SDS Page

    4) Product Images from "Regulation of ErbB2 Receptor Status by the Proteasomal DUB POH1"

    Article Title: Regulation of ErbB2 Receptor Status by the Proteasomal DUB POH1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005544

    Multiple POH1 oligos down-regulate ErbB2. A. siRNA mediated knockdown (KD) of candidates was repeated with four individual On Target Plus oligos incubated with HeLa cells for 48 hours. The results for POH1 were consistent. The knockdown of other candidates (MYSM1, USP14, BAP1, etc) with four individual oligos failed to show significant effects on ErbB2 levels (results from MYSM1 shown as an example). Lysates were also probed with EGFR, Met, and tubulin antibodies. The effect of knock-down of POH1 on EGFR and Met levels was much less pronounced than for ErbB2. B. quantitation of ErbB2, EGFR, and Met receptors for POH1 knockdown cells (averaged from 3 experiments). C. HeLa cells were treated with POH1 siRNA (four individual oligos) or oligofectamine transfection reagent (control) for 48 hours before lysis and analysed by immunoblotting for Transferrin receptor (TfR), Hrs, STAM, AMSH, and tubulin. Knock-down of POH1 showed minor effects on the levels of these proteins.
    Figure Legend Snippet: Multiple POH1 oligos down-regulate ErbB2. A. siRNA mediated knockdown (KD) of candidates was repeated with four individual On Target Plus oligos incubated with HeLa cells for 48 hours. The results for POH1 were consistent. The knockdown of other candidates (MYSM1, USP14, BAP1, etc) with four individual oligos failed to show significant effects on ErbB2 levels (results from MYSM1 shown as an example). Lysates were also probed with EGFR, Met, and tubulin antibodies. The effect of knock-down of POH1 on EGFR and Met levels was much less pronounced than for ErbB2. B. quantitation of ErbB2, EGFR, and Met receptors for POH1 knockdown cells (averaged from 3 experiments). C. HeLa cells were treated with POH1 siRNA (four individual oligos) or oligofectamine transfection reagent (control) for 48 hours before lysis and analysed by immunoblotting for Transferrin receptor (TfR), Hrs, STAM, AMSH, and tubulin. Knock-down of POH1 showed minor effects on the levels of these proteins.

    Techniques Used: Incubation, Quantitation Assay, Transfection, Lysis

    Effects of ErbB2 depletion on EGFR down-regulation and downstream signaling in HeLa cells. A. HeLa cells were treated with ErbB2 siRNA or oligofectamine transfection reagent (control) for 48 hours before stimulation with 100 ng/ml EGF for various time periods. Lysates were analysed by immunoblotting with EGFR and ErbB2 antibodies. B. quantitation of A showing EGFR down-regulation was not significantly affected (data averaged from 3 experiments). C. HeLa cells were treated as in A and incubated with 10 ng/ml EGF for different time periods and lysed. Lysate was analysed by immunoblotting with ErbB2, pMAPK, MAPK, and tubulin antibodies. Levels of total MAPK and pMAPK were not affected by ErbB2 knock-down.
    Figure Legend Snippet: Effects of ErbB2 depletion on EGFR down-regulation and downstream signaling in HeLa cells. A. HeLa cells were treated with ErbB2 siRNA or oligofectamine transfection reagent (control) for 48 hours before stimulation with 100 ng/ml EGF for various time periods. Lysates were analysed by immunoblotting with EGFR and ErbB2 antibodies. B. quantitation of A showing EGFR down-regulation was not significantly affected (data averaged from 3 experiments). C. HeLa cells were treated as in A and incubated with 10 ng/ml EGF for different time periods and lysed. Lysate was analysed by immunoblotting with ErbB2, pMAPK, MAPK, and tubulin antibodies. Levels of total MAPK and pMAPK were not affected by ErbB2 knock-down.

    Techniques Used: Transfection, Quantitation Assay, Incubation

    POH1 depletion and ErbB2 receptor turnover. A, HeLa cells were treated±POH1 siRNA for 48 hours before incubation with 10 µg/ml cycloheximide. Cells were lysed and analysed by immunoblotting with ErbB2 29D8 and Ab20 antibodies, which recognize intracellular and extracellular epitopes of ErbB2 respectively, EGFR, and tubulin antibodies. B, quantitation shows that both EGFR (by antibody 1005) and ErbB2 (by antibodies Ab20 and 29D8) are turned over more rapidly in POH1 knock-down cells (data averaged from 3 experiments). C. HeLa cells were treated with four On Target Plus oligos (POH1) or with oligofectamine alone for 72 hours before lysis with hot lysis buffer. A higher molecular weight ErbB2 “smear” was observed in all 4 knock-down samples. D The high molecular weight smear associated with ErbB2 immuno-reactivity is sensitive to treatment with a deubiquitinase (USP2). HeLa cells were treated with POH1 siRNA or oligofectamine for 48 hours before lysis in the presence of NEM. ErbB2 was immunoprecipitated and treated in vitro with USP2 catalytic domain (100 nM, 8 hours, 37°C). Samples were analyzed by immunoblotting with ErbB2 antibodies targeting extracellular (Ab20) and intracellular (29D8) domains. Note that the smear detected with Ab20 is lost upon USP2 treatment whilst detection with the intracellular domain antibody increases. As a control for USP2 DUB-activity, EGFR was immunoprecipitated from EGF-stimulated (5 min) HeLa cells and treated in vitro with USP2 catalytic domain before SDS-PAGE and western blotting with anti-Ubiquitin.
    Figure Legend Snippet: POH1 depletion and ErbB2 receptor turnover. A, HeLa cells were treated±POH1 siRNA for 48 hours before incubation with 10 µg/ml cycloheximide. Cells were lysed and analysed by immunoblotting with ErbB2 29D8 and Ab20 antibodies, which recognize intracellular and extracellular epitopes of ErbB2 respectively, EGFR, and tubulin antibodies. B, quantitation shows that both EGFR (by antibody 1005) and ErbB2 (by antibodies Ab20 and 29D8) are turned over more rapidly in POH1 knock-down cells (data averaged from 3 experiments). C. HeLa cells were treated with four On Target Plus oligos (POH1) or with oligofectamine alone for 72 hours before lysis with hot lysis buffer. A higher molecular weight ErbB2 “smear” was observed in all 4 knock-down samples. D The high molecular weight smear associated with ErbB2 immuno-reactivity is sensitive to treatment with a deubiquitinase (USP2). HeLa cells were treated with POH1 siRNA or oligofectamine for 48 hours before lysis in the presence of NEM. ErbB2 was immunoprecipitated and treated in vitro with USP2 catalytic domain (100 nM, 8 hours, 37°C). Samples were analyzed by immunoblotting with ErbB2 antibodies targeting extracellular (Ab20) and intracellular (29D8) domains. Note that the smear detected with Ab20 is lost upon USP2 treatment whilst detection with the intracellular domain antibody increases. As a control for USP2 DUB-activity, EGFR was immunoprecipitated from EGF-stimulated (5 min) HeLa cells and treated in vitro with USP2 catalytic domain before SDS-PAGE and western blotting with anti-Ubiquitin.

    Techniques Used: Incubation, Quantitation Assay, Lysis, Molecular Weight, Immunoprecipitation, In Vitro, Activity Assay, SDS Page, Western Blot

    Chronic treatment with a proteasome inhibitor replicates effects of POH1 depletion on ErbB2. HeLa cells were incubated with epoxomicin (8 nM or 10 nM) or DMSO for 48 hours (fresh inhibitors were applied at 24 hours). Cell lysates obtained by “hot lysis” were subjected to SDS-PAGE followed by immunoblotting with ErbB2, EGFR, TrfR, POH1, and tubulin antibodies. Epoxomicin treatment resulted in the apparent loss of ErbB2 and concomitant appearance of a high molecular weight smear.
    Figure Legend Snippet: Chronic treatment with a proteasome inhibitor replicates effects of POH1 depletion on ErbB2. HeLa cells were incubated with epoxomicin (8 nM or 10 nM) or DMSO for 48 hours (fresh inhibitors were applied at 24 hours). Cell lysates obtained by “hot lysis” were subjected to SDS-PAGE followed by immunoblotting with ErbB2, EGFR, TrfR, POH1, and tubulin antibodies. Epoxomicin treatment resulted in the apparent loss of ErbB2 and concomitant appearance of a high molecular weight smear.

    Techniques Used: Incubation, Lysis, SDS Page, Molecular Weight

    ErbB2 escapes EGF induced down-regulation. A, Comparison of ErbB2 receptor levels in HeLa and SKBr3 cells. Cell lysate samples corresponding to the indicated number of cells were separated by SDS-PAGE and immunoblotted with ErbB2 antibodies and IR800-coupled secondary antibodies. The relative amount of ErbB2 per cell was calculated based on Odyssey scans as discussed in Materials and Methods . B, HeLa, HEK293T (H293T), A549, and DU145 cells were stimulated with 100 ng/ml EGF for 2 hours and lysed in parallel with unstimulated cells. The lysate was subjected to SDS-PAGE and immunoblotting with EGFR, ErbB2, and tubulin antibodies. EGFR is down-regulated after 2 hours stimulation, but ErbB2 remains stable. C, HeLa and SKBr3 cells were treated with 100 ng/ml EGF for 1 or 2 hours and analysed by immunoblotting with EGFR and ErbB2 antibodies. No EGFR was detected in SKBr3 cells. Note that EGFR antibody (2232) cross-reacts with high levels of ErbB2 in SKBr3 cells.
    Figure Legend Snippet: ErbB2 escapes EGF induced down-regulation. A, Comparison of ErbB2 receptor levels in HeLa and SKBr3 cells. Cell lysate samples corresponding to the indicated number of cells were separated by SDS-PAGE and immunoblotted with ErbB2 antibodies and IR800-coupled secondary antibodies. The relative amount of ErbB2 per cell was calculated based on Odyssey scans as discussed in Materials and Methods . B, HeLa, HEK293T (H293T), A549, and DU145 cells were stimulated with 100 ng/ml EGF for 2 hours and lysed in parallel with unstimulated cells. The lysate was subjected to SDS-PAGE and immunoblotting with EGFR, ErbB2, and tubulin antibodies. EGFR is down-regulated after 2 hours stimulation, but ErbB2 remains stable. C, HeLa and SKBr3 cells were treated with 100 ng/ml EGF for 1 or 2 hours and analysed by immunoblotting with EGFR and ErbB2 antibodies. No EGFR was detected in SKBr3 cells. Note that EGFR antibody (2232) cross-reacts with high levels of ErbB2 in SKBr3 cells.

    Techniques Used: SDS Page

    POH1 depletion does not differentially affect ErbB2 and EGFR transcription levels. HeLa cells were treated±POH1 siRNA (two individual oligos) for 24 or 48 hours (left and right panels respectively) before RNA was extracted. Levels of mRNA of EGFR and ErbB2 were calculated relative to actin mRNA. Graph shows qRT-PCR results averaged from 3 experiments. Error bars show standard deviation.
    Figure Legend Snippet: POH1 depletion does not differentially affect ErbB2 and EGFR transcription levels. HeLa cells were treated±POH1 siRNA (two individual oligos) for 24 or 48 hours (left and right panels respectively) before RNA was extracted. Levels of mRNA of EGFR and ErbB2 were calculated relative to actin mRNA. Graph shows qRT-PCR results averaged from 3 experiments. Error bars show standard deviation.

    Techniques Used: Quantitative RT-PCR, Standard Deviation

    Cell surface levels of ErbB2 are relatively insensitive to POH1 knock-down. A, HeLa cells were treated with ErbB2, POH1 siRNA or oligofectamine alone for 48 hours before detachment with 2 mM EDTA. One million cells from each condition were labelled with FITC-conjugated ErbB2 and phycoerythrin (PE) conjugated EGFR antibodies and then analysed by flow cytometry. B, quantification of A shows relative amounts of fluorescence for each condition. C, samples from A were analysed by immunoblotting with ErbB2 (Ab20 and 29D8), EGFR, POH1, and tubulin antibodies.
    Figure Legend Snippet: Cell surface levels of ErbB2 are relatively insensitive to POH1 knock-down. A, HeLa cells were treated with ErbB2, POH1 siRNA or oligofectamine alone for 48 hours before detachment with 2 mM EDTA. One million cells from each condition were labelled with FITC-conjugated ErbB2 and phycoerythrin (PE) conjugated EGFR antibodies and then analysed by flow cytometry. B, quantification of A shows relative amounts of fluorescence for each condition. C, samples from A were analysed by immunoblotting with ErbB2 (Ab20 and 29D8), EGFR, POH1, and tubulin antibodies.

    Techniques Used: Flow Cytometry, Cytometry, Fluorescence

    5) Product Images from "The transient receptor potential type vanilloid 1 suppresses skin carcinogenesis"

    Article Title: The transient receptor potential type vanilloid 1 suppresses skin carcinogenesis

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-08-3263

    TRPV1 suppresses EGFR signaling and cell transformation. ( A , left) PCR detection of genomic TRPV1 in TRPV1 wildtype (WT) and knockout (KO) mouse skin. ( A , right) RT-PCR detection of TRPV1 in wildtype (TRPV1 +/+ ) and KO (TRPV1 −/− ) mouse
    Figure Legend Snippet: TRPV1 suppresses EGFR signaling and cell transformation. ( A , left) PCR detection of genomic TRPV1 in TRPV1 wildtype (WT) and knockout (KO) mouse skin. ( A , right) RT-PCR detection of TRPV1 in wildtype (TRPV1 +/+ ) and KO (TRPV1 −/− ) mouse

    Techniques Used: Transformation Assay, Polymerase Chain Reaction, Knock-Out, Reverse Transcription Polymerase Chain Reaction

    The TRPV1 and EGFR interact in vitro and ex vivo . ( A ) GST-fusion protein constructs were created as follows: TRPV1-FL (full length TRPV1); GST-TRPV1-NT (N-terminal cytoplasmic domain of TRPV1); and GST-TRPV1-CT (C-terminal cytoplasmic domain of TRPV1).
    Figure Legend Snippet: The TRPV1 and EGFR interact in vitro and ex vivo . ( A ) GST-fusion protein constructs were created as follows: TRPV1-FL (full length TRPV1); GST-TRPV1-NT (N-terminal cytoplasmic domain of TRPV1); and GST-TRPV1-CT (C-terminal cytoplasmic domain of TRPV1).

    Techniques Used: In Vitro, Ex Vivo, Construct

    The TRPV1 induces ubiquitylation and degradation of the EGFR protein through the lysosomal pathway. ( A ) A431 human skin cells were transfected with increasing amounts of TRPV1 and total and phosphorylated (Tyr1068) levels of EGFR and TRPV1 proteins were
    Figure Legend Snippet: The TRPV1 induces ubiquitylation and degradation of the EGFR protein through the lysosomal pathway. ( A ) A431 human skin cells were transfected with increasing amounts of TRPV1 and total and phosphorylated (Tyr1068) levels of EGFR and TRPV1 proteins were

    Techniques Used: Transfection

    TRPV1 interacts with the Cbl protein for the down-regulation of the EGFR. (A) A431 human skin cells were combined with GST-mock, GST-TRPV1-NT, or GST-TRPV1-CT. A GST pull down assay was performed and Cbl was detected by Western blot analysis. (B) A431
    Figure Legend Snippet: TRPV1 interacts with the Cbl protein for the down-regulation of the EGFR. (A) A431 human skin cells were combined with GST-mock, GST-TRPV1-NT, or GST-TRPV1-CT. A GST pull down assay was performed and Cbl was detected by Western blot analysis. (B) A431

    Techniques Used: Pull Down Assay, Western Blot

    TRPV1 and EGFR are expressed in opposition to each other. ( A ) EGFR-deficient (EGFR −/− ) MEFs express high levels of endogenous TRPV1 compared to wildtype (EGFR +/+ ) cells. ( B ) Ectopic expression of the TRPV1 suppresses the endogenous level
    Figure Legend Snippet: TRPV1 and EGFR are expressed in opposition to each other. ( A ) EGFR-deficient (EGFR −/− ) MEFs express high levels of endogenous TRPV1 compared to wildtype (EGFR +/+ ) cells. ( B ) Ectopic expression of the TRPV1 suppresses the endogenous level

    Techniques Used: Expressing

    6) Product Images from "Role for engagement of β‐arrestin2 by the transactivated EGFR in agonist‐specific regulation of δ receptor activation of ERK1/2) Role for engagement of β‐arrestin2 by the transactivated EGFR in agonist‐specific regulation of δ receptor activation of ERK1/2"

    Article Title: Role for engagement of β‐arrestin2 by the transactivated EGFR in agonist‐specific regulation of δ receptor activation of ERK1/2) Role for engagement of β‐arrestin2 by the transactivated EGFR in agonist‐specific regulation of δ receptor activation of ERK1/2

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.13254

    Schematic representation of δ receptor‐mediated ERK1/2 activation via the PKCδ/EGFR/β‐arrestin2 signalling pathway in response to specific agonist stimulation. DPDPE binding to δ receptors results in the
    Figure Legend Snippet: Schematic representation of δ receptor‐mediated ERK1/2 activation via the PKCδ/EGFR/β‐arrestin2 signalling pathway in response to specific agonist stimulation. DPDPE binding to δ receptors results in the

    Techniques Used: Activation Assay, Binding Assay

    PKCδ acts upstream of metalloproteinase activation and is required for the release of the EGFR‐activating factor. (A) Serum‐starved δ receptor‐null receiving cells previously treated or not for 30 min with
    Figure Legend Snippet: PKCδ acts upstream of metalloproteinase activation and is required for the release of the EGFR‐activating factor. (A) Serum‐starved δ receptor‐null receiving cells previously treated or not for 30 min with

    Techniques Used: Activation Assay

    β‐Arrestin2 is required for DPDPE‐stimulated ERK1/2 activation and acts downstream of the PKCδ‐mediated activation of metalloproteinase and transactivation of EGFR in HEK293 cells stably expressing the S363A δ
    Figure Legend Snippet: β‐Arrestin2 is required for DPDPE‐stimulated ERK1/2 activation and acts downstream of the PKCδ‐mediated activation of metalloproteinase and transactivation of EGFR in HEK293 cells stably expressing the S363A δ

    Techniques Used: Activation Assay, Stable Transfection, Expressing

    δ Receptor‐mediated ERK1/2 activation stimulated by TIPP or morphine but not by DPDPE involves the transactivation of EGFR in cultured cells and native tissues. (A, B) Serum‐starved HEK293 δ receptor cells or NG108‐15
    Figure Legend Snippet: δ Receptor‐mediated ERK1/2 activation stimulated by TIPP or morphine but not by DPDPE involves the transactivation of EGFR in cultured cells and native tissues. (A, B) Serum‐starved HEK293 δ receptor cells or NG108‐15

    Techniques Used: Activation Assay, Cell Culture

    β‐Arrestin2 is required for δ receptor‐mediated ERK1/2 activation stimulated by morphine and TIPP but not by DPDPE and acts downstream of EGFR transactivation. (A) HEK293 δ receptor cells were transfected with siRNAs
    Figure Legend Snippet: β‐Arrestin2 is required for δ receptor‐mediated ERK1/2 activation stimulated by morphine and TIPP but not by DPDPE and acts downstream of EGFR transactivation. (A) HEK293 δ receptor cells were transfected with siRNAs

    Techniques Used: Activation Assay, Transfection

    DPDPE can activate ERK1/2 by employing β‐arrestin2 via PKCδ‐mediated transactivation of EGFR in HEK293 cells expressing the S363A mutant δ receptor. (A) Cells stably expressing wild‐type δ receptors
    Figure Legend Snippet: DPDPE can activate ERK1/2 by employing β‐arrestin2 via PKCδ‐mediated transactivation of EGFR in HEK293 cells expressing the S363A mutant δ receptor. (A) Cells stably expressing wild‐type δ receptors

    Techniques Used: Expressing, Mutagenesis, Stable Transfection

    PKCδ activity is required for δ receptor‐mediated ERK1/2 activation via transactivation of EGFR in response to TIPP or morphine but not DPDPE stimulation. (A) Serum‐starved HEK293 δ receptor cells were pretreated
    Figure Legend Snippet: PKCδ activity is required for δ receptor‐mediated ERK1/2 activation via transactivation of EGFR in response to TIPP or morphine but not DPDPE stimulation. (A) Serum‐starved HEK293 δ receptor cells were pretreated

    Techniques Used: Activity Assay, Activation Assay

    7) Product Images from "Hepatocellular alterations and dysregulation of oncogenic pathways in the liver of transgenic mice overexpressing growth hormone"

    Article Title: Hepatocellular alterations and dysregulation of oncogenic pathways in the liver of transgenic mice overexpressing growth hormone

    Journal: Cell Cycle

    doi: 10.4161/cc.24026

    Figure 3. Expression and phosphorylation of STAT5, STAT3, Akt, mTOR and EGFR in the liver of GH-overexpressing transgenic mice and normal controls. Liver extracts from young adult normal female (NF), normal male (NM), GH-transgenic female (TF)
    Figure Legend Snippet: Figure 3. Expression and phosphorylation of STAT5, STAT3, Akt, mTOR and EGFR in the liver of GH-overexpressing transgenic mice and normal controls. Liver extracts from young adult normal female (NF), normal male (NM), GH-transgenic female (TF)

    Techniques Used: Expressing, Transgenic Assay, Mouse Assay

    8) Product Images from "Expression of oncogenic K-ras and loss of Smad4 cooperate to induce the expression of EGFR and to promote invasion of immortalized human pancreas ductal cells"

    Article Title: Expression of oncogenic K-ras and loss of Smad4 cooperate to induce the expression of EGFR and to promote invasion of immortalized human pancreas ductal cells

    Journal: International journal of cancer. Journal international du cancer

    doi: 10.1002/ijc.25412

    Increased invasion of HPNE cells modified by Smad4 knock down or expression of oncogenic ras requires EGFR signaling
    Figure Legend Snippet: Increased invasion of HPNE cells modified by Smad4 knock down or expression of oncogenic ras requires EGFR signaling

    Techniques Used: Modification, Expressing

    Oncogenic K-ras increases EGFR protein stability and up regulates EGFR transcription and Smad4-dependent TGF-β signaling suppresses EGFR transcription
    Figure Legend Snippet: Oncogenic K-ras increases EGFR protein stability and up regulates EGFR transcription and Smad4-dependent TGF-β signaling suppresses EGFR transcription

    Techniques Used:

    Expression of oncogenic K-ras and knock down of Smad4 cooperate to induce EGFR and erbB2 expression in immortalized HPNE cells
    Figure Legend Snippet: Expression of oncogenic K-ras and knock down of Smad4 cooperate to induce EGFR and erbB2 expression in immortalized HPNE cells

    Techniques Used: Expressing

    9) Product Images from "Glycerol kinase 5 confers gefitinib resistance through SREBP1/SCD1 signaling pathway"

    Article Title: Glycerol kinase 5 confers gefitinib resistance through SREBP1/SCD1 signaling pathway

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-019-1057-7

    GK5 knockdown suppresses PC9R cell proliferation by inhibiting EGFR/AKT/SREBP1/SCD1 signaling molecules. a Whole human cDNA array analysis. b Western blotting on protein levels of EGFR, p-AKT, AKT, survivin, PLK1, SREBP1, SCD1 and Bcl-2 in PC9R cells c CCK8 assay on cell proliferation of PC9R cells overexpressing EGFR and infected with viral particles expressing shGK5–1 and shNEG. d CCK8 assay on cell proliferation of PC9R cells transfected with shGK5–1, − 2 or shNEG and treated with YM155. * p
    Figure Legend Snippet: GK5 knockdown suppresses PC9R cell proliferation by inhibiting EGFR/AKT/SREBP1/SCD1 signaling molecules. a Whole human cDNA array analysis. b Western blotting on protein levels of EGFR, p-AKT, AKT, survivin, PLK1, SREBP1, SCD1 and Bcl-2 in PC9R cells c CCK8 assay on cell proliferation of PC9R cells overexpressing EGFR and infected with viral particles expressing shGK5–1 and shNEG. d CCK8 assay on cell proliferation of PC9R cells transfected with shGK5–1, − 2 or shNEG and treated with YM155. * p

    Techniques Used: Western Blot, CCK-8 Assay, Infection, Expressing, Transfection

    GK5 mediates gefitinib resistance. a Total Internal Reflection Fluorescence (TIRF) Microscopy images of exosomes isolated from plasma of EGFR TKI-sensitive and -resistant patients. b Exosomal GK5 mRNA in the plasma of gefitinib-sensitive and -resistant lung adenocarcinoma patients measured using TCLN biochips. c , d The RT-PCR and Western blotting on GK5 expression in gefitinib-sensitive PC9 and gefitinib-resistant PC9R cells. * p
    Figure Legend Snippet: GK5 mediates gefitinib resistance. a Total Internal Reflection Fluorescence (TIRF) Microscopy images of exosomes isolated from plasma of EGFR TKI-sensitive and -resistant patients. b Exosomal GK5 mRNA in the plasma of gefitinib-sensitive and -resistant lung adenocarcinoma patients measured using TCLN biochips. c , d The RT-PCR and Western blotting on GK5 expression in gefitinib-sensitive PC9 and gefitinib-resistant PC9R cells. * p

    Techniques Used: Fluorescence, Microscopy, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing

    10) Product Images from "Ion channel TRPV1-dependent activation of PTP1B suppresses EGFR-associated intestinal tumorigenesis"

    Article Title: Ion channel TRPV1-dependent activation of PTP1B suppresses EGFR-associated intestinal tumorigenesis

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI72340

    TRPV1 regulates EGFR activity through calpain and PTP1B. ( A ) HCT116 cells were transfected with control or TRPV1 plasmid, then pretreated with DMSO or ALLM (10 μM) for 1 hour, followed by EGF (1 ng/ml) stimulation. Total cell lysates were analyzed by Western blotting. Lanes were run on the same gel. ( B ) HCT116 cells transfected with control or TRPV1 plasmid were pretreated with DMSO, the nonselective PTP inhibitor Na 3 VO 4 (10 μM), or the selective PTP1B inhibitor Compound 3 (10 μM) for 1 hour, followed by EGF stimulation and Western blotting. ( C ) Q-PCR was performed for PTP candidates with RNA isolated from WT colon crypts. ( D ) HCT116 cells were treated with control or PTP1B siRNA and transfected with control or TRPV1 plasmid. Cells were then stimulated with EGF (1 ng/ml) and analyzed by Western blotting. Representative results of 2 independent experiments. ( E ) HCT116 cells were transfected with empty vector, WT PTP1B (PTP435), or PTP1B C-terminal truncation mutants (PTP370 and PTP377). Cells were stimulated with EGF (1 ng/ml) and analyzed by Western blotting. Representative results of 3 independent experiments. ( F ) PTP1B expression in intestinal organoids, shown by confocal fluorescent microscopy. ( G ) Expression of PTP1B in murine small intestinal tissue sections. Immunostaining for PTP1B and detection with DAB (brown) was followed by counterstaining with hematoxylin. ( H ) Q-PCR analysis of villus (V1 and V2) and crypt (C) fractions for Trpv1 and Ptpn1 , showing enrichment in the latter. Values are expressed relative to the V1 fraction. Mean ± SEM ( n = 3 per group). * P
    Figure Legend Snippet: TRPV1 regulates EGFR activity through calpain and PTP1B. ( A ) HCT116 cells were transfected with control or TRPV1 plasmid, then pretreated with DMSO or ALLM (10 μM) for 1 hour, followed by EGF (1 ng/ml) stimulation. Total cell lysates were analyzed by Western blotting. Lanes were run on the same gel. ( B ) HCT116 cells transfected with control or TRPV1 plasmid were pretreated with DMSO, the nonselective PTP inhibitor Na 3 VO 4 (10 μM), or the selective PTP1B inhibitor Compound 3 (10 μM) for 1 hour, followed by EGF stimulation and Western blotting. ( C ) Q-PCR was performed for PTP candidates with RNA isolated from WT colon crypts. ( D ) HCT116 cells were treated with control or PTP1B siRNA and transfected with control or TRPV1 plasmid. Cells were then stimulated with EGF (1 ng/ml) and analyzed by Western blotting. Representative results of 2 independent experiments. ( E ) HCT116 cells were transfected with empty vector, WT PTP1B (PTP435), or PTP1B C-terminal truncation mutants (PTP370 and PTP377). Cells were stimulated with EGF (1 ng/ml) and analyzed by Western blotting. Representative results of 3 independent experiments. ( F ) PTP1B expression in intestinal organoids, shown by confocal fluorescent microscopy. ( G ) Expression of PTP1B in murine small intestinal tissue sections. Immunostaining for PTP1B and detection with DAB (brown) was followed by counterstaining with hematoxylin. ( H ) Q-PCR analysis of villus (V1 and V2) and crypt (C) fractions for Trpv1 and Ptpn1 , showing enrichment in the latter. Values are expressed relative to the V1 fraction. Mean ± SEM ( n = 3 per group). * P

    Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Western Blot, Polymerase Chain Reaction, Isolation, Expressing, Microscopy, Immunostaining

    Proposed model for EGFR-TRPV1 crosstalk in IECs. TRPV1 and PTP1B are part of a homeostatic signaling circuit that restrains EGFR-induced epithelial cell proliferation. EGFR kinase activity mediates proproliferative and thus protumorigenic effects in IECs (dashed red arrows). TRPV1 and PTP1B are predominantly expressed in the crypt compartment that contains TA cells (dark blue cells) with active EGFR signaling. Ligand-induced autophosphorylation of the EGFR results in PLC activation, which cleaves PIP 2 , a tonic inhibitor of TRPV1, into diacylglycerol (DAG) and inositol triphosphate (IP 3 ; green arrows). This results in TRPV1 triggering and Ca 2+ influx, which activates calpain and subsequently PTP1B. PTP1B then dephosphorylates EGFR (blue lines). This coupling between EGFR and TRPV1 exerts negative feedback on growth factor receptor signaling, inhibits crypt progenitor cell (dark blue cells) turnover, and hence reduces the risk of intestinal neoplasia development (red cells). +4 ISC, ISC at +4 position; CBCC, crypt-based columnar stem cell.
    Figure Legend Snippet: Proposed model for EGFR-TRPV1 crosstalk in IECs. TRPV1 and PTP1B are part of a homeostatic signaling circuit that restrains EGFR-induced epithelial cell proliferation. EGFR kinase activity mediates proproliferative and thus protumorigenic effects in IECs (dashed red arrows). TRPV1 and PTP1B are predominantly expressed in the crypt compartment that contains TA cells (dark blue cells) with active EGFR signaling. Ligand-induced autophosphorylation of the EGFR results in PLC activation, which cleaves PIP 2 , a tonic inhibitor of TRPV1, into diacylglycerol (DAG) and inositol triphosphate (IP 3 ; green arrows). This results in TRPV1 triggering and Ca 2+ influx, which activates calpain and subsequently PTP1B. PTP1B then dephosphorylates EGFR (blue lines). This coupling between EGFR and TRPV1 exerts negative feedback on growth factor receptor signaling, inhibits crypt progenitor cell (dark blue cells) turnover, and hence reduces the risk of intestinal neoplasia development (red cells). +4 ISC, ISC at +4 position; CBCC, crypt-based columnar stem cell.

    Techniques Used: Activity Assay, Planar Chromatography, Activation Assay

    TRPV1 signaling inhibits cell proliferation and EGFR activity in vivo. ( A ) Increased IEC proliferation in colons from naive Trpv1 –/– versus WT mice, as shown by Ki67 immunostaining. ( B ) Increased EGFR Y1068 phosphorylation in Trpv1 –/– IECs was reversed by gefitinib treatment. Mice were treated with 2 doses of gefitinib (50 mg/kg, gavage) with a 6-hour interval, followed by IEC harvesting and Western blotting. Shown are representative results from 3 experiments. ( C ) Immunostaining for p-EGFR Y1068 of distal colon tissues. ( D ) Quantification with ImageJ. 3 representative areas were used for analysis, with 3 mice per group. ( E ) Hyperproliferation of Trpv1 –/– IECs was reversed by EGFR inhibition with gefitinib (50 mg/kg) for 5 consecutive days before tissue harvesting. ( F ) Quantification of Ki67 + cells by ImageJ (ImmunoRatio plugin). ( G ) Capsaicin administration suppressed transcription of EGFR target genes in a TRPV1-dependent manner, comparable to gefitinib. Mice received DMSO, capsaicin (3 mg/kg), gefitinib (50 mg/kg), or capsaicin plus gefitinib by gavage, and IECs were prepared 6 hours later. Shown is Q-PCR analysis for c-Fos or Fosl2 , relative to the respective DMSO control. ( H ) Q-PCR analysis for c-Jun . ( I ) Generation of transgenic TRPV1 IEC mice. ( J ) TRPV1 fl/fl and TRPV1 IEC mice were analyzed for p-EGFR Y1068 by Western blotting. IEC-specific TRPV1 overexpression inhibited constitutive EGFR signaling in IECs. ( K ) IEC-specific TRPV1 overexpression suppressed EGF-induced EGFR Y1068 phosphorylation in IECs. Data are mean ± SEM ( D and F ; n = 3 per group) or mean ± SD ( G and H ; n = 2 per group). * P
    Figure Legend Snippet: TRPV1 signaling inhibits cell proliferation and EGFR activity in vivo. ( A ) Increased IEC proliferation in colons from naive Trpv1 –/– versus WT mice, as shown by Ki67 immunostaining. ( B ) Increased EGFR Y1068 phosphorylation in Trpv1 –/– IECs was reversed by gefitinib treatment. Mice were treated with 2 doses of gefitinib (50 mg/kg, gavage) with a 6-hour interval, followed by IEC harvesting and Western blotting. Shown are representative results from 3 experiments. ( C ) Immunostaining for p-EGFR Y1068 of distal colon tissues. ( D ) Quantification with ImageJ. 3 representative areas were used for analysis, with 3 mice per group. ( E ) Hyperproliferation of Trpv1 –/– IECs was reversed by EGFR inhibition with gefitinib (50 mg/kg) for 5 consecutive days before tissue harvesting. ( F ) Quantification of Ki67 + cells by ImageJ (ImmunoRatio plugin). ( G ) Capsaicin administration suppressed transcription of EGFR target genes in a TRPV1-dependent manner, comparable to gefitinib. Mice received DMSO, capsaicin (3 mg/kg), gefitinib (50 mg/kg), or capsaicin plus gefitinib by gavage, and IECs were prepared 6 hours later. Shown is Q-PCR analysis for c-Fos or Fosl2 , relative to the respective DMSO control. ( H ) Q-PCR analysis for c-Jun . ( I ) Generation of transgenic TRPV1 IEC mice. ( J ) TRPV1 fl/fl and TRPV1 IEC mice were analyzed for p-EGFR Y1068 by Western blotting. IEC-specific TRPV1 overexpression inhibited constitutive EGFR signaling in IECs. ( K ) IEC-specific TRPV1 overexpression suppressed EGF-induced EGFR Y1068 phosphorylation in IECs. Data are mean ± SEM ( D and F ; n = 3 per group) or mean ± SD ( G and H ; n = 2 per group). * P

    Techniques Used: Activity Assay, In Vivo, Mouse Assay, Immunostaining, Western Blot, Inhibition, Polymerase Chain Reaction, Transgenic Assay, Over Expression

    11) Product Images from "Multiplexed Targeting of Barrett’s Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft In Vivo and Human Specimens Ex Vivo"

    Article Title: Multiplexed Targeting of Barrett’s Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft In Vivo and Human Specimens Ex Vivo

    Journal: Journal of medicinal chemistry

    doi: 10.1021/acs.jmedchem.8b00405

    No effect of peptide heterodimer on cell signaling. We evaluated the effect of the QRH*-KSP*-E3-Cy5.5 on downstream cell signaling after binding to SKBr3 cells. On Western blot, we observed no change in phosphorylation of EGFR (p-EGFR), ErbB2 (p-ErbB2) or of downstream AKT (p-AKT) and ERK (p-ERK) with incubation of heterodimer at 1, 5, and 20 μM. By comparison, the addition of EGF, an endogenous ligand for EGFR, showed increased expression of p-AKT and p-ERK. The addition of 100 nM of lapatinib, a tyrosine kinase inhibitor known to interrupt EGFR/ErbB2 signaling in solid tumors, showed reduced expression of p-EGFR, p-ErbB2 and p-AKT. Cells treated with 1% DMSO and untreated cells showed no suppression of EGFR and ErbB2 mediated signaling. β-tubulin is used as loading control.
    Figure Legend Snippet: No effect of peptide heterodimer on cell signaling. We evaluated the effect of the QRH*-KSP*-E3-Cy5.5 on downstream cell signaling after binding to SKBr3 cells. On Western blot, we observed no change in phosphorylation of EGFR (p-EGFR), ErbB2 (p-ErbB2) or of downstream AKT (p-AKT) and ERK (p-ERK) with incubation of heterodimer at 1, 5, and 20 μM. By comparison, the addition of EGF, an endogenous ligand for EGFR, showed increased expression of p-AKT and p-ERK. The addition of 100 nM of lapatinib, a tyrosine kinase inhibitor known to interrupt EGFR/ErbB2 signaling in solid tumors, showed reduced expression of p-EGFR, p-ErbB2 and p-AKT. Cells treated with 1% DMSO and untreated cells showed no suppression of EGFR and ErbB2 mediated signaling. β-tubulin is used as loading control.

    Techniques Used: Binding Assay, Western Blot, Incubation, Expressing

    Characterization of peptide heterodimer. In siRNA knockdown experiments, QRH*-KSP*-E3-Cy5.5 (red) shows significantly greater binding to the surface (arrows) of A) siCL (control) SkBr3 cells compared with that for B) siEGFR (knockdown) cells. Similar results were found for C) siCL and D) siErbB2 (knockdown) cells. E) Quantified results show significantly greater intensity for siCL versus siEGFR and siCL versus siErbB2, P =3.6×10 −4 and P =7.8×10 −3 , respectively, by unpaired t-test. The mean value was calculated from 5 cells chosen randomly from 3 images collected independently. F) Western blot shows EGFR and ErbB2 expression in control and knockdown cells. G) The apparent dissociation constant (binding affinity) for QRH*-KSP*-E3-Cy5.5 was found to be k d = 23 versus 98 and 54 nM for QRH*-Cy5.5 and KSP*-Cy5.5. H) The apparent association time constant for QRH*-KSP*-E3-Cy5.5 was found to be k = 0.22 min −1 (4.5 min) versus 0.21 min −1 (4.8 min) and 0.35 min −1 (2.9 min) for QRH*-Cy5.5 and KSP*-Cy5.5. Results for each measurement are representative of 3 independent experiments.
    Figure Legend Snippet: Characterization of peptide heterodimer. In siRNA knockdown experiments, QRH*-KSP*-E3-Cy5.5 (red) shows significantly greater binding to the surface (arrows) of A) siCL (control) SkBr3 cells compared with that for B) siEGFR (knockdown) cells. Similar results were found for C) siCL and D) siErbB2 (knockdown) cells. E) Quantified results show significantly greater intensity for siCL versus siEGFR and siCL versus siErbB2, P =3.6×10 −4 and P =7.8×10 −3 , respectively, by unpaired t-test. The mean value was calculated from 5 cells chosen randomly from 3 images collected independently. F) Western blot shows EGFR and ErbB2 expression in control and knockdown cells. G) The apparent dissociation constant (binding affinity) for QRH*-KSP*-E3-Cy5.5 was found to be k d = 23 versus 98 and 54 nM for QRH*-Cy5.5 and KSP*-Cy5.5. H) The apparent association time constant for QRH*-KSP*-E3-Cy5.5 was found to be k = 0.22 min −1 (4.5 min) versus 0.21 min −1 (4.8 min) and 0.35 min −1 (2.9 min) for QRH*-Cy5.5 and KSP*-Cy5.5. Results for each measurement are representative of 3 independent experiments.

    Techniques Used: Binding Assay, Western Blot, Expressing

    Optimization of peptide heterodimer. On confocal microscopy, fluorescence from binding of candidate heterodimer with linkers A ) E2, B ) Hex, C ) E3, D ) E6, and E ) E10 to the surface (arrow) of SKBr3 cells can be seen. F ) Quantified results show that the E3 linker provides the highest mean fluorescence intensity. P -values were determined using unpaired t-test. Measurements are an average of 10 randomly chosen cells from 4 images collected independently. G ) Western blot shows EGFR and ErbB2 expression in SKBr3 and QhTERT cells.
    Figure Legend Snippet: Optimization of peptide heterodimer. On confocal microscopy, fluorescence from binding of candidate heterodimer with linkers A ) E2, B ) Hex, C ) E3, D ) E6, and E ) E10 to the surface (arrow) of SKBr3 cells can be seen. F ) Quantified results show that the E3 linker provides the highest mean fluorescence intensity. P -values were determined using unpaired t-test. Measurements are an average of 10 randomly chosen cells from 4 images collected independently. G ) Western blot shows EGFR and ErbB2 expression in SKBr3 and QhTERT cells.

    Techniques Used: Confocal Microscopy, Fluorescence, Binding Assay, Western Blot, Expressing

    Specific binding of peptide heterodimer to Barrett’s neoplasia. On representative confocal microscopy images of human esophageal specimens ex vivo, QRH*-KSP*-E3-Cy5.5 (red) shows minimal staining to A) squamous (SQ) and B) Barrett’s esophagus (BE), and increased intensity with C) high-grade dysplasia (HGD) and D) esophageal adenocarcinoma (EAC). Similar results were found with AF568-labeled anti-EGFR antibody (yellow) and for AF488-labeled anti-ErbB2 antibody (green). Merged images show co-localization of peptide and antibody binding.
    Figure Legend Snippet: Specific binding of peptide heterodimer to Barrett’s neoplasia. On representative confocal microscopy images of human esophageal specimens ex vivo, QRH*-KSP*-E3-Cy5.5 (red) shows minimal staining to A) squamous (SQ) and B) Barrett’s esophagus (BE), and increased intensity with C) high-grade dysplasia (HGD) and D) esophageal adenocarcinoma (EAC). Similar results were found with AF568-labeled anti-EGFR antibody (yellow) and for AF488-labeled anti-ErbB2 antibody (green). Merged images show co-localization of peptide and antibody binding.

    Techniques Used: Binding Assay, Confocal Microscopy, Ex Vivo, Staining, Labeling

    Co-localization of peptide heterodimer and antibody binding to Barrett’s neoplasia. A) On confocal microscopy, serial sections of HGD in human esophageal specimens are shown following staining with QRH*-KSP*-E3-Cy5.5 (red), anti-EGFR antibody labeled with AF568 (yellow) and anti-ErbB2 antibody labeled with AF488 (green). Fluorescence intensities were quantified from the mean of a set of 3 boxes with dimensions of 20×20 μm 2 placed over random crypts. Co-localization of binding can be appreciated on the merged image. B) High-magnification images are shown from dashed boxes. On the merged image, Pearson’s correlation coefficient of ρ = 0.60 and 0.75 was measured for EGFR and ErbB2, respectively. C) From n = 31, 8, 23, and 12 specimens of SQ, BE, HGD, and EAC, respectively, we found significantly greater mean fluorescence intensity from HGD and EAC compared with that for BE and SQ with QRH*-KSP*-E3-Cy5.5, the P -value for difference are calculated by Tukey’s multiple comparisons. A similar result was found for anti-EGFR-AF568 and anti-ErbB2-AF488. D) ROC curve shows 88% sensitivity, 87% specificity and 0.95 AUC with QRH*-KSP*-E3-Cy5.5; 74% sensitivity, 69% specificity, and 0.79 AUC with QRH*-Cy5.5, and 85% sensitivity, 79% specificity, and 0.91 AUC with KSP*-Cy5.5.
    Figure Legend Snippet: Co-localization of peptide heterodimer and antibody binding to Barrett’s neoplasia. A) On confocal microscopy, serial sections of HGD in human esophageal specimens are shown following staining with QRH*-KSP*-E3-Cy5.5 (red), anti-EGFR antibody labeled with AF568 (yellow) and anti-ErbB2 antibody labeled with AF488 (green). Fluorescence intensities were quantified from the mean of a set of 3 boxes with dimensions of 20×20 μm 2 placed over random crypts. Co-localization of binding can be appreciated on the merged image. B) High-magnification images are shown from dashed boxes. On the merged image, Pearson’s correlation coefficient of ρ = 0.60 and 0.75 was measured for EGFR and ErbB2, respectively. C) From n = 31, 8, 23, and 12 specimens of SQ, BE, HGD, and EAC, respectively, we found significantly greater mean fluorescence intensity from HGD and EAC compared with that for BE and SQ with QRH*-KSP*-E3-Cy5.5, the P -value for difference are calculated by Tukey’s multiple comparisons. A similar result was found for anti-EGFR-AF568 and anti-ErbB2-AF488. D) ROC curve shows 88% sensitivity, 87% specificity and 0.95 AUC with QRH*-KSP*-E3-Cy5.5; 74% sensitivity, 69% specificity, and 0.79 AUC with QRH*-Cy5.5, and 85% sensitivity, 79% specificity, and 0.91 AUC with KSP*-Cy5.5.

    Techniques Used: Binding Assay, Confocal Microscopy, Staining, Labeling, Fluorescence

    12) Product Images from "Pegylated siRNA-loaded calcium phosphate nanoparticle-driven amplification of cancer cell internalization in vivo"

    Article Title: Pegylated siRNA-loaded calcium phosphate nanoparticle-driven amplification of cancer cell internalization in vivo

    Journal: Biomaterials

    doi: 10.1016/j.biomaterials.2013.01.046

    Doxorubicin-incorporated nanoparticles enhance siRNA delivery and target-specific knock-down in cancer cells. (a) A decrease in relative mRNA expression of XIAP, PARP, VEGF, and EGFR in H292 cells following a 24 h treatment with corresponding nanoparticles.
    Figure Legend Snippet: Doxorubicin-incorporated nanoparticles enhance siRNA delivery and target-specific knock-down in cancer cells. (a) A decrease in relative mRNA expression of XIAP, PARP, VEGF, and EGFR in H292 cells following a 24 h treatment with corresponding nanoparticles.

    Techniques Used: Expressing

    13) Product Images from "The bile acid receptor GPBAR1 (TGR5) is expressed in human gastric cancers and promotes epithelial-mesenchymal transition in gastric cancer cell lines"

    Article Title: The bile acid receptor GPBAR1 (TGR5) is expressed in human gastric cancers and promotes epithelial-mesenchymal transition in gastric cancer cell lines

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10477

    GPBAR1 activation by TLCA in MKN45 cells causes EGFR phosphorylation A. Cells treated with 100 μM TLCA were harvested at the indicated time. Cell lysates were immunoblotted with antibodies against phosphorylated EGFR (pEGFR) and total EGFR as indicated. B. Quantitative densitometry of the protein expression of the phosphorylated EGFR and total form. *
    Figure Legend Snippet: GPBAR1 activation by TLCA in MKN45 cells causes EGFR phosphorylation A. Cells treated with 100 μM TLCA were harvested at the indicated time. Cell lysates were immunoblotted with antibodies against phosphorylated EGFR (pEGFR) and total EGFR as indicated. B. Quantitative densitometry of the protein expression of the phosphorylated EGFR and total form. *

    Techniques Used: Activation Assay, Expressing

    14) Product Images from "Oxidative Stress-Induced circHBEGF Promotes Extracellular Matrix Production via Regulating miR-646/EGFR in Human Trabecular Meshwork Cells"

    Article Title: Oxidative Stress-Induced circHBEGF Promotes Extracellular Matrix Production via Regulating miR-646/EGFR in Human Trabecular Meshwork Cells

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2020/4692034

    Proposed working model based on our studies. Schematic summarizing our proposed model for circHBEGF in promoting ECM production in HTMCs. circHBEGF directly adsorb miR-646 as a sponge, through which regulate the expression of EGFR. Therefore, circHBEGF can activate EGF signaling (STAT3, AKT, and ERK), through which transcriptionally upregulates ECM gene expression in HTMCs.
    Figure Legend Snippet: Proposed working model based on our studies. Schematic summarizing our proposed model for circHBEGF in promoting ECM production in HTMCs. circHBEGF directly adsorb miR-646 as a sponge, through which regulate the expression of EGFR. Therefore, circHBEGF can activate EGF signaling (STAT3, AKT, and ERK), through which transcriptionally upregulates ECM gene expression in HTMCs.

    Techniques Used: Expressing

    circHBEGF promotes ECM genes through EGF signaling. (a) Western blot analysis of EGFR and ECM genes in HTMCs after EGFR knockdown. (b, d) Western blot analysis of p-STAT3, p-AKT and p-ERK, in HTMCs transfected with the indicated condition. (c, e) Quantification of p-STAT3, p-AKT, and p-ERK related to total protein in (b) and (d).
    Figure Legend Snippet: circHBEGF promotes ECM genes through EGF signaling. (a) Western blot analysis of EGFR and ECM genes in HTMCs after EGFR knockdown. (b, d) Western blot analysis of p-STAT3, p-AKT and p-ERK, in HTMCs transfected with the indicated condition. (c, e) Quantification of p-STAT3, p-AKT, and p-ERK related to total protein in (b) and (d).

    Techniques Used: Western Blot, Transfection

    15) Product Images from "The endogenous subcellular localisations of the long chain fatty acid-activating enzymes ACSL3 and ACSL4 in sarcoma and breast cancer cells"

    Article Title: The endogenous subcellular localisations of the long chain fatty acid-activating enzymes ACSL3 and ACSL4 in sarcoma and breast cancer cells

    Journal: Molecular and Cellular Biochemistry

    doi: 10.1007/s11010-018-3332-x

    Equilibrium distributions of organelle marker proteins, ACSL3 and ACSL4 in HT1080 sucrose density gradient fractions. a Equal volumes of all HT1080 subcellular fractions were subjected to SDS-PAGE separation and immunoblotted for the ER marker protein calnexin; the TGN-endosomal protein syntaxin-6; the late endosomal protein LAMP1; the plasma membrane-associated EGFR, the early endosome-recruited protein EEA1; the lysosomal protein prenylcysteine lyase and the mitochondrial protein mitofusin-1, ACSL3 and ACSL4, and the inositol phospholipid-dependent enzymes Akt, PLCγ and PI4KIIα. Data presented are representative of experiments repeated 3–4 times with similar results. b The relative normalised distributions of anti-ACSL3 and anti-ACSL4 immunoreactivities in the gradient fractions. Western blotting signals were quantified using imageJ software. Data are representitive of experiments repeated 3–4 times with similar results
    Figure Legend Snippet: Equilibrium distributions of organelle marker proteins, ACSL3 and ACSL4 in HT1080 sucrose density gradient fractions. a Equal volumes of all HT1080 subcellular fractions were subjected to SDS-PAGE separation and immunoblotted for the ER marker protein calnexin; the TGN-endosomal protein syntaxin-6; the late endosomal protein LAMP1; the plasma membrane-associated EGFR, the early endosome-recruited protein EEA1; the lysosomal protein prenylcysteine lyase and the mitochondrial protein mitofusin-1, ACSL3 and ACSL4, and the inositol phospholipid-dependent enzymes Akt, PLCγ and PI4KIIα. Data presented are representative of experiments repeated 3–4 times with similar results. b The relative normalised distributions of anti-ACSL3 and anti-ACSL4 immunoreactivities in the gradient fractions. Western blotting signals were quantified using imageJ software. Data are representitive of experiments repeated 3–4 times with similar results

    Techniques Used: Marker, SDS Page, Western Blot, Software

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    Immunoprecipitation:

    Article Title: Glucose deprivation activates a metabolic and signaling amplification loop leading to cell death
    Article Snippet: .. Antibodies and reagents Antibodies used for western blotting and immunoprecipitation included: anti-phospho-tyrosine (clone 4G10) from Upstate; anti-phospho-ERK1/2, anti-phospho-JNK, anti-phospho-p38alpha, anti-phospho-S235/236-S6, anti-phospho-S473-Akt, anti-phospho-Y1045-EGFR, anti-phospho-Y1068-EGFR, anti-total EGFR, anti-phospho-Y1349 Met, anti-phospho-Y1234/Y1235-Met, anti-phospho-751-PDGFRβ, anti-phospho-Y416-SRC, and anti-phospho-Y397-FAK from Cell Signaling Technology; anti-PTEN from Cascade Biosciences; anti-total FAK from BD Biosciences; anti-total GRB2 and anti-total PTP-1B from Santa Cruz Biotechnology. .. Catalase and pNPP were purchased from Sigma.

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    Cell Signaling Technology Inc site specific phospho egfr antibodies
    BTC induces less <t>EGFR</t> phosphorylation than EGF in hTCEpi and MDA-MB-468 cells. (A and B) Cells were treated with 1.6 nM EGF or 1.6 nM BTC for the indicated times. Cell lysates were prepared and immunoblotted with the indicated EGFR phosphotyrosine-specific antibodies (pY998, <t>pY1045,</t> pY1068, pY1148), total EGFR, or α -tubulin. (A) hTCEpi cell representative blots from three experiments. (B) MDA-MB-468 cell representative blots from three experiments.
    Site Specific Phospho Egfr Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti egfr
    <t>EGFR</t> and <t>DNAPKcs</t> cellular localisation following IR treatment. NIH3T3 transiently transfected with wtEGFR, NLS123, L858R, LNLS123, EGFRvIII, M1, M12, KMT, ΔNLS and Vector control were serum starved for 24 hours, treated with 4Gy Ionising radiation and then fixed with 4%PFA 20 minutes following treatment. Cells were stained with Goat anti-Rabbit Alexa fluor 647 (EGFR), Goat anti-Mouse Alexa fluor 488 (DNAPKcs), and Dapi (nucleus).
    Anti Egfr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc egfr
    Deregulated ERK signalling in metaplastic epithelial cells . A . Paired cultures of epithelial cells (EC-85) were analyzed by immunoblotting for cytokine-mediated phosphorylation of <t>STAT1,</t> STAT3 and ERK. Basal level of phosphorylated ERK but not of phosphorylated STATs is elevated. B , Expression of <t>EGFR,</t> STAT3 and ERK in the untreated cultures was determined by immunoblotting. C , Cultures of normal and metaplastic cells were treated for 3 h with serum-free RPMI containing 0.1% carrier DMSO or the same medium with 10 μM U0126. The level of phosphorylated ERK was determined by immunoblotting. D , DNA synthesis was determined in response to treatment with serially diluted OSM and conditioned medium of LPS activated macrophages. One set of cultures of normal and metaplastic cells were also treated for 3 hour with 10 μM U0126 in normal growth medium prior to the addition of [ 3 H]thymidine. The incorporation of [ 3 H]thymidine (mean and range of duplicate cultures) were normalized to the number of cells and expressed relative to the values determined for the control cultures of the normal epithelial cells.
    Egfr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BTC induces less EGFR phosphorylation than EGF in hTCEpi and MDA-MB-468 cells. (A and B) Cells were treated with 1.6 nM EGF or 1.6 nM BTC for the indicated times. Cell lysates were prepared and immunoblotted with the indicated EGFR phosphotyrosine-specific antibodies (pY998, pY1045, pY1068, pY1148), total EGFR, or α -tubulin. (A) hTCEpi cell representative blots from three experiments. (B) MDA-MB-468 cell representative blots from three experiments.

    Journal: Molecular Pharmacology

    Article Title: Betacellulin (BTC) Biases the EGFR To Dimerize with ErbB3

    doi: 10.1124/mol.118.113399

    Figure Lengend Snippet: BTC induces less EGFR phosphorylation than EGF in hTCEpi and MDA-MB-468 cells. (A and B) Cells were treated with 1.6 nM EGF or 1.6 nM BTC for the indicated times. Cell lysates were prepared and immunoblotted with the indicated EGFR phosphotyrosine-specific antibodies (pY998, pY1045, pY1068, pY1148), total EGFR, or α -tubulin. (A) hTCEpi cell representative blots from three experiments. (B) MDA-MB-468 cell representative blots from three experiments.

    Article Snippet: The indicated proteins were immunoblotted using the following antibodies: EGFR (SC-03) and EGFR (A-10) antibodies obtained from Santa Cruz Biotechnology (Dallas, TX), site-specific phospho-EGFR antibodies (pY998, pY1045, pY1068, and pY1148) were from Cell Signaling Technology (Danvers, MA), and α -tubulin antibodies were from Sigma-Aldrich.

    Techniques: Multiple Displacement Amplification

    EGFR and DNAPKcs cellular localisation following IR treatment. NIH3T3 transiently transfected with wtEGFR, NLS123, L858R, LNLS123, EGFRvIII, M1, M12, KMT, ΔNLS and Vector control were serum starved for 24 hours, treated with 4Gy Ionising radiation and then fixed with 4%PFA 20 minutes following treatment. Cells were stained with Goat anti-Rabbit Alexa fluor 647 (EGFR), Goat anti-Mouse Alexa fluor 488 (DNAPKcs), and Dapi (nucleus).

    Journal: Cancer research

    Article Title: EGFR NUCLEAR TRANSLOCATION MODULATES DNA REPAIR FOLLOWING CISPLATIN AND IONIZING RADIATION TREATMENT

    doi: 10.1158/0008-5472.CAN-10-2384

    Figure Lengend Snippet: EGFR and DNAPKcs cellular localisation following IR treatment. NIH3T3 transiently transfected with wtEGFR, NLS123, L858R, LNLS123, EGFRvIII, M1, M12, KMT, ΔNLS and Vector control were serum starved for 24 hours, treated with 4Gy Ionising radiation and then fixed with 4%PFA 20 minutes following treatment. Cells were stained with Goat anti-Rabbit Alexa fluor 647 (EGFR), Goat anti-Mouse Alexa fluor 488 (DNAPKcs), and Dapi (nucleus).

    Article Snippet: Proteins were probed using anti-EGFR (Cell Signalling 1:1000), anti-DNAPKcs (AbCam 1:400) anti-PY20 (Santa Cruz 1:1000).

    Techniques: Transfection, Plasmid Preparation, Staining

    (A) EGFR-DNAPKcs complex cellular localization. Stable NIH3T3 cells expressing wtEGFR, NLS123, EGFRvIII and Vector control were treated with 50μM cisplatin for one hour in serum free media and then fixed with 4%PFA 18 hours following treatment or 4 Gy and then fixed with 4%PFA 20 minutes follwowing radiation. Cells were then immuno blocked with anti-rabbit EGFR and anti-mouse DNAPKcs. Interacting complexes were then visulised via the duo link proximit assay. Each red spot represents a single interaction. (B) EGFR modulation of DNA-PK kinase activity. Stable NIH3T3 cells expressing wtEGFR, NLS123, L858R, LNLS123 and EGFRvIII were treated with 50μM cisplatin for one hour or 4gy or 100ng/ml EGF in serum free media. 18 hours following the treatment with cisplatin, 20 minutes following the treatment with IR and at 1 hour following EGF incubation samples prepared for the DNAPK Kinase assay. The graph shows the percentage change in DNAPK activity following each treatment compared to untreated. Stars indicate statistical significance.

    Journal: Cancer research

    Article Title: EGFR NUCLEAR TRANSLOCATION MODULATES DNA REPAIR FOLLOWING CISPLATIN AND IONIZING RADIATION TREATMENT

    doi: 10.1158/0008-5472.CAN-10-2384

    Figure Lengend Snippet: (A) EGFR-DNAPKcs complex cellular localization. Stable NIH3T3 cells expressing wtEGFR, NLS123, EGFRvIII and Vector control were treated with 50μM cisplatin for one hour in serum free media and then fixed with 4%PFA 18 hours following treatment or 4 Gy and then fixed with 4%PFA 20 minutes follwowing radiation. Cells were then immuno blocked with anti-rabbit EGFR and anti-mouse DNAPKcs. Interacting complexes were then visulised via the duo link proximit assay. Each red spot represents a single interaction. (B) EGFR modulation of DNA-PK kinase activity. Stable NIH3T3 cells expressing wtEGFR, NLS123, L858R, LNLS123 and EGFRvIII were treated with 50μM cisplatin for one hour or 4gy or 100ng/ml EGF in serum free media. 18 hours following the treatment with cisplatin, 20 minutes following the treatment with IR and at 1 hour following EGF incubation samples prepared for the DNAPK Kinase assay. The graph shows the percentage change in DNAPK activity following each treatment compared to untreated. Stars indicate statistical significance.

    Article Snippet: Proteins were probed using anti-EGFR (Cell Signalling 1:1000), anti-DNAPKcs (AbCam 1:400) anti-PY20 (Santa Cruz 1:1000).

    Techniques: Expressing, Plasmid Preparation, Activity Assay, Incubation, Kinase Assay

    (A) wtEGFR Transfected NI3T3 cells were treated with 50μM cisplatin for 1 hour in serum free media. Cells were then lysed 1,9,12,15,18,24, hours following treatment. 750μg of protein lysate were immunoprecipitated using anti DNAPKcs and blotted with anti EGFR and anti DNAPKcs. EGFR pull-down was quantified by 2D densitometric analysis and shown as a binding fold compared to the untreated control. Mouse unrelated antibody (M IGg) was used as negative control. (B) Stable NIH3T3 cells expressing wtEGFR, NLS123, L858R, LNLS123, EGFRvIII, and Vector control were treated with 50μM cisplatin or 4 G IR, or treated with 100ng/ml EGF as described in the materials and methods. 1.5mg of protein lysate was then immunoprecipitated using anti-EGFR monoclonal antibody and blotted with anti DNAPKcs, anti-PY20 and anti-EGFR.

    Journal: Cancer research

    Article Title: EGFR NUCLEAR TRANSLOCATION MODULATES DNA REPAIR FOLLOWING CISPLATIN AND IONIZING RADIATION TREATMENT

    doi: 10.1158/0008-5472.CAN-10-2384

    Figure Lengend Snippet: (A) wtEGFR Transfected NI3T3 cells were treated with 50μM cisplatin for 1 hour in serum free media. Cells were then lysed 1,9,12,15,18,24, hours following treatment. 750μg of protein lysate were immunoprecipitated using anti DNAPKcs and blotted with anti EGFR and anti DNAPKcs. EGFR pull-down was quantified by 2D densitometric analysis and shown as a binding fold compared to the untreated control. Mouse unrelated antibody (M IGg) was used as negative control. (B) Stable NIH3T3 cells expressing wtEGFR, NLS123, L858R, LNLS123, EGFRvIII, and Vector control were treated with 50μM cisplatin or 4 G IR, or treated with 100ng/ml EGF as described in the materials and methods. 1.5mg of protein lysate was then immunoprecipitated using anti-EGFR monoclonal antibody and blotted with anti DNAPKcs, anti-PY20 and anti-EGFR.

    Article Snippet: Proteins were probed using anti-EGFR (Cell Signalling 1:1000), anti-DNAPKcs (AbCam 1:400) anti-PY20 (Santa Cruz 1:1000).

    Techniques: Transfection, Immunoprecipitation, Binding Assay, Negative Control, Expressing, Plasmid Preparation

    EGFR and DNAPKcs cellular localisation following cisplatin treatment. NIH3T3 transiently transfected with wtEGFR, NLS123, L858R, LNLS123, EGFRvIII, M1, M12, KMT, ΔNLS and Vector control, were treated with 50μM cisplatin for one hour in serum free media and then fixed with 4%PFA 18 hours following treatment. Cells were stained with Goat anti-Rabbit Alexa fluor 647 (EGFR), Goat anti-Mouse Alexa fluor 488 (DNAPKcs), and Dapi (nucleus).

    Journal: Cancer research

    Article Title: EGFR NUCLEAR TRANSLOCATION MODULATES DNA REPAIR FOLLOWING CISPLATIN AND IONIZING RADIATION TREATMENT

    doi: 10.1158/0008-5472.CAN-10-2384

    Figure Lengend Snippet: EGFR and DNAPKcs cellular localisation following cisplatin treatment. NIH3T3 transiently transfected with wtEGFR, NLS123, L858R, LNLS123, EGFRvIII, M1, M12, KMT, ΔNLS and Vector control, were treated with 50μM cisplatin for one hour in serum free media and then fixed with 4%PFA 18 hours following treatment. Cells were stained with Goat anti-Rabbit Alexa fluor 647 (EGFR), Goat anti-Mouse Alexa fluor 488 (DNAPKcs), and Dapi (nucleus).

    Article Snippet: Proteins were probed using anti-EGFR (Cell Signalling 1:1000), anti-DNAPKcs (AbCam 1:400) anti-PY20 (Santa Cruz 1:1000).

    Techniques: Transfection, Plasmid Preparation, Staining

    EGFR and DNAPKcs cellular localisation following cisplatin treatment. Stable NIH3T3 cells expressing wtEGFR, NL123, L858R, LNLS123, EGFRvIII, Vector control were treated with 50μM cisplatin for one hour in serum free media and then fixed with 4%PFA 18 hours following treatment. Cells were stained with Goat anti-Rabbit Alexa fluor 647 (EGFR), Goat anti-Mouse Alexa fluor 488 (DNAPKcs), and Dapi (nucleus).

    Journal: Cancer research

    Article Title: EGFR NUCLEAR TRANSLOCATION MODULATES DNA REPAIR FOLLOWING CISPLATIN AND IONIZING RADIATION TREATMENT

    doi: 10.1158/0008-5472.CAN-10-2384

    Figure Lengend Snippet: EGFR and DNAPKcs cellular localisation following cisplatin treatment. Stable NIH3T3 cells expressing wtEGFR, NL123, L858R, LNLS123, EGFRvIII, Vector control were treated with 50μM cisplatin for one hour in serum free media and then fixed with 4%PFA 18 hours following treatment. Cells were stained with Goat anti-Rabbit Alexa fluor 647 (EGFR), Goat anti-Mouse Alexa fluor 488 (DNAPKcs), and Dapi (nucleus).

    Article Snippet: Proteins were probed using anti-EGFR (Cell Signalling 1:1000), anti-DNAPKcs (AbCam 1:400) anti-PY20 (Santa Cruz 1:1000).

    Techniques: Expressing, Plasmid Preparation, Staining

    EGFR and DNAPKcs cellular localisation following IR treatment. Stable NIH3T3 cells expressing wtEGFR, NL123, L858R, LNLS123, EGFRvIII, Vector control were serum starved for 24 hours, treated with 4gy Ionising radiation and then fixed with 4%PFA 20 minutes following treatment. Cells were stained with Goat anti-Rabbit Alexa fluor 647 (EGFR), Goat anti-Mouse Alexa fluor 488 (DNAPKcs), and Dapi (nucleus).

    Journal: Cancer research

    Article Title: EGFR NUCLEAR TRANSLOCATION MODULATES DNA REPAIR FOLLOWING CISPLATIN AND IONIZING RADIATION TREATMENT

    doi: 10.1158/0008-5472.CAN-10-2384

    Figure Lengend Snippet: EGFR and DNAPKcs cellular localisation following IR treatment. Stable NIH3T3 cells expressing wtEGFR, NL123, L858R, LNLS123, EGFRvIII, Vector control were serum starved for 24 hours, treated with 4gy Ionising radiation and then fixed with 4%PFA 20 minutes following treatment. Cells were stained with Goat anti-Rabbit Alexa fluor 647 (EGFR), Goat anti-Mouse Alexa fluor 488 (DNAPKcs), and Dapi (nucleus).

    Article Snippet: Proteins were probed using anti-EGFR (Cell Signalling 1:1000), anti-DNAPKcs (AbCam 1:400) anti-PY20 (Santa Cruz 1:1000).

    Techniques: Expressing, Plasmid Preparation, Staining

    Chromatin immunoprecipitation assay and Western blotting analysis of c-Myc, iNOS, Cyclin D1, and VEGF expression in Panc-1 and Colo-357 cells. (A), Agarose gel electrophoresis of the Polymerase Chain Reaction (PCR)-amplified c-Myc gene fragment from the chromatin DNA precipitated with antibody against EGFR, Src, or Stat3, or with the non-specific IgG; and (B and C), Immunoblotting analysis of whole-cell lysates probing for EGFR or Src (B(i) and C(i)) or c-Myc, iNOS, Cyclin D1 or VEGF (B(ii) and C(ii)), and the effects of siRNA knockdown of EGFR (EGFR siRNA), Src (Src siRNA) or control (con) siRNA, or S3I-201 or Das). Bands corresponding to proteins or c-Myc gene in gel are shown; M, molecular weight marker, EGFR/Src, sequential immunoprecipitation with anti-EGFR and then anti-Src antibody. Data are representative of 3 independent studies, and values are mean and s.d of 3 independent studies; * p -

    Journal: PLoS ONE

    Article Title: A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0019605

    Figure Lengend Snippet: Chromatin immunoprecipitation assay and Western blotting analysis of c-Myc, iNOS, Cyclin D1, and VEGF expression in Panc-1 and Colo-357 cells. (A), Agarose gel electrophoresis of the Polymerase Chain Reaction (PCR)-amplified c-Myc gene fragment from the chromatin DNA precipitated with antibody against EGFR, Src, or Stat3, or with the non-specific IgG; and (B and C), Immunoblotting analysis of whole-cell lysates probing for EGFR or Src (B(i) and C(i)) or c-Myc, iNOS, Cyclin D1 or VEGF (B(ii) and C(ii)), and the effects of siRNA knockdown of EGFR (EGFR siRNA), Src (Src siRNA) or control (con) siRNA, or S3I-201 or Das). Bands corresponding to proteins or c-Myc gene in gel are shown; M, molecular weight marker, EGFR/Src, sequential immunoprecipitation with anti-EGFR and then anti-Src antibody. Data are representative of 3 independent studies, and values are mean and s.d of 3 independent studies; * p -

    Article Snippet: A smaller size increase was observed from anti-EGFR antibody ( , anti-EGFR) and the rabbit IgG ( , IgG).

    Techniques: Chromatin Immunoprecipitation, Western Blot, Expressing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Molecular Weight, Marker, Immunoprecipitation

    Co-immunoprecipitation with immunoblotting analysis of EGFR, Src and Stat3 complex in the nucleus and the sub-cellular distribution of EGFR, Src and Stat3. (A and B) Immunoblotting analyses of immunecomplexes of EGFR (IP:EGFR), Src (IP:Src), Stat3 (IP:Stat3), EGFR/Src (IP:EGFR/IP:Src), or of non-specific IgG non-immuneprecpitate prepared from nuclear extracts of Panc-1 or Colo-357 cells and probing for Stat3, EGFR, Src, or the Tata-binding protein (TBP); and (C), immunoblotting analysis of membrane (mem) and cytosolic (cyto) fractions and of nuclear (nuc) extracts from Panc-1 cells probing for (i) EGFR, (ii) Stat3 and (iii) Src. Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of nuclear extract used in the assay; IP:EGFR/IP:Src, sequential immunoprecipitation with anti-EGFR and then anti-Src antibody; Data are representative of 3 independent studies.

    Journal: PLoS ONE

    Article Title: A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0019605

    Figure Lengend Snippet: Co-immunoprecipitation with immunoblotting analysis of EGFR, Src and Stat3 complex in the nucleus and the sub-cellular distribution of EGFR, Src and Stat3. (A and B) Immunoblotting analyses of immunecomplexes of EGFR (IP:EGFR), Src (IP:Src), Stat3 (IP:Stat3), EGFR/Src (IP:EGFR/IP:Src), or of non-specific IgG non-immuneprecpitate prepared from nuclear extracts of Panc-1 or Colo-357 cells and probing for Stat3, EGFR, Src, or the Tata-binding protein (TBP); and (C), immunoblotting analysis of membrane (mem) and cytosolic (cyto) fractions and of nuclear (nuc) extracts from Panc-1 cells probing for (i) EGFR, (ii) Stat3 and (iii) Src. Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of nuclear extract used in the assay; IP:EGFR/IP:Src, sequential immunoprecipitation with anti-EGFR and then anti-Src antibody; Data are representative of 3 independent studies.

    Article Snippet: A smaller size increase was observed from anti-EGFR antibody ( , anti-EGFR) and the rabbit IgG ( , IgG).

    Techniques: Immunoprecipitation, Binding Assay

    Studies of protein complex and protein binding partners using the Detection and Analysis through Nanoparticle Sizing technology. (A) Kinetic binding assay of EGFR-gold nanoparticle (GNP) probe (or mouse IgG1-GNP probe as negative control) binding to (i) EGFR protein and its complex from Panc-1 nuclear extracts, and the (ii) inhibitory effect of the mouse monoclonal anti-EGFR antibody on the EGFR-GNP probe binding to the EGFR protein; and (B) Protein complex binding partner analysis whereby the polyclonal anti-Stat3, anti-Src or anti-EGFR antibody or the non-specific rabbit IgG (negative control) is added to the assay solution prepared from the (i) non-specific mouse IgG1-GNP probe (negative control), or (ii) anti-EGFR-GNP probe; Data are representative of 4 independent studies.

    Journal: PLoS ONE

    Article Title: A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0019605

    Figure Lengend Snippet: Studies of protein complex and protein binding partners using the Detection and Analysis through Nanoparticle Sizing technology. (A) Kinetic binding assay of EGFR-gold nanoparticle (GNP) probe (or mouse IgG1-GNP probe as negative control) binding to (i) EGFR protein and its complex from Panc-1 nuclear extracts, and the (ii) inhibitory effect of the mouse monoclonal anti-EGFR antibody on the EGFR-GNP probe binding to the EGFR protein; and (B) Protein complex binding partner analysis whereby the polyclonal anti-Stat3, anti-Src or anti-EGFR antibody or the non-specific rabbit IgG (negative control) is added to the assay solution prepared from the (i) non-specific mouse IgG1-GNP probe (negative control), or (ii) anti-EGFR-GNP probe; Data are representative of 4 independent studies.

    Article Snippet: A smaller size increase was observed from anti-EGFR antibody ( , anti-EGFR) and the rabbit IgG ( , IgG).

    Techniques: Protein Binding, Binding Assay, Negative Control

    Co-immunoprecipitation with immunoblotting analysis of EGFR, Src and Stat3 association in Panc-1 and Colo-357 cells. Immunoblotting analyses of immunecomplexes of EGFR (IP:EGFR), Src (IP:Src), and Stat3 (IP:Stat3), or of non-specific IgG non-immunoprecipitate prepared from whole-cell lysates of Panc-1 or Colo-357 cells untransfected (A and B) or transfected with EGFR siRNA, Src siRNA, or control (con) siRNA (C) and probing for Src, Stat3 and EGFR in the absence (A and C) or presence (B) of Stat3 blocking peptide (Stat3 BP), Src blocking peptide (Src BP) or EGFR blocking peptide (EGFR BP). Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of lysate used in the assay; Data are representative of 3 independent studies.

    Journal: PLoS ONE

    Article Title: A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0019605

    Figure Lengend Snippet: Co-immunoprecipitation with immunoblotting analysis of EGFR, Src and Stat3 association in Panc-1 and Colo-357 cells. Immunoblotting analyses of immunecomplexes of EGFR (IP:EGFR), Src (IP:Src), and Stat3 (IP:Stat3), or of non-specific IgG non-immunoprecipitate prepared from whole-cell lysates of Panc-1 or Colo-357 cells untransfected (A and B) or transfected with EGFR siRNA, Src siRNA, or control (con) siRNA (C) and probing for Src, Stat3 and EGFR in the absence (A and C) or presence (B) of Stat3 blocking peptide (Stat3 BP), Src blocking peptide (Src BP) or EGFR blocking peptide (EGFR BP). Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of lysate used in the assay; Data are representative of 3 independent studies.

    Article Snippet: A smaller size increase was observed from anti-EGFR antibody ( , anti-EGFR) and the rabbit IgG ( , IgG).

    Techniques: Immunoprecipitation, Transfection, Blocking Assay

    Deregulated ERK signalling in metaplastic epithelial cells . A . Paired cultures of epithelial cells (EC-85) were analyzed by immunoblotting for cytokine-mediated phosphorylation of STAT1, STAT3 and ERK. Basal level of phosphorylated ERK but not of phosphorylated STATs is elevated. B , Expression of EGFR, STAT3 and ERK in the untreated cultures was determined by immunoblotting. C , Cultures of normal and metaplastic cells were treated for 3 h with serum-free RPMI containing 0.1% carrier DMSO or the same medium with 10 μM U0126. The level of phosphorylated ERK was determined by immunoblotting. D , DNA synthesis was determined in response to treatment with serially diluted OSM and conditioned medium of LPS activated macrophages. One set of cultures of normal and metaplastic cells were also treated for 3 hour with 10 μM U0126 in normal growth medium prior to the addition of [ 3 H]thymidine. The incorporation of [ 3 H]thymidine (mean and range of duplicate cultures) were normalized to the number of cells and expressed relative to the values determined for the control cultures of the normal epithelial cells.

    Journal: BMC Cancer

    Article Title: Transformation of human bronchial epithelial cells alters responsiveness to inflammatory cytokines

    doi: 10.1186/1471-2407-5-145

    Figure Lengend Snippet: Deregulated ERK signalling in metaplastic epithelial cells . A . Paired cultures of epithelial cells (EC-85) were analyzed by immunoblotting for cytokine-mediated phosphorylation of STAT1, STAT3 and ERK. Basal level of phosphorylated ERK but not of phosphorylated STATs is elevated. B , Expression of EGFR, STAT3 and ERK in the untreated cultures was determined by immunoblotting. C , Cultures of normal and metaplastic cells were treated for 3 h with serum-free RPMI containing 0.1% carrier DMSO or the same medium with 10 μM U0126. The level of phosphorylated ERK was determined by immunoblotting. D , DNA synthesis was determined in response to treatment with serially diluted OSM and conditioned medium of LPS activated macrophages. One set of cultures of normal and metaplastic cells were also treated for 3 hour with 10 μM U0126 in normal growth medium prior to the addition of [ 3 H]thymidine. The incorporation of [ 3 H]thymidine (mean and range of duplicate cultures) were normalized to the number of cells and expressed relative to the values determined for the control cultures of the normal epithelial cells.

    Article Snippet: The membranes were reacted with antibodies to phospho-specific forms or ERK1/2, STAT1, STAT3 and EGFR (Cell Signalling Technology, Inc., Beverly, MA) and total forms of ERK1/2, STAT1, and STAT3, LIFR (Santa Cruz, Santa Cruz, CA).

    Techniques: Expressing, DNA Synthesis

    Cytokine-specific signalling in bronchial epithelial cells . Paired primary cultures of normal and metaplastic epithelial cells (EC-14) were treated for 15 min with the factors listed at the bottom. The relative levels of phosphorylated STAT1, STAT3, EGFR and ERK, as well as the total STAT3 and ERK, were determined by immunoblotting.

    Journal: BMC Cancer

    Article Title: Transformation of human bronchial epithelial cells alters responsiveness to inflammatory cytokines

    doi: 10.1186/1471-2407-5-145

    Figure Lengend Snippet: Cytokine-specific signalling in bronchial epithelial cells . Paired primary cultures of normal and metaplastic epithelial cells (EC-14) were treated for 15 min with the factors listed at the bottom. The relative levels of phosphorylated STAT1, STAT3, EGFR and ERK, as well as the total STAT3 and ERK, were determined by immunoblotting.

    Article Snippet: The membranes were reacted with antibodies to phospho-specific forms or ERK1/2, STAT1, STAT3 and EGFR (Cell Signalling Technology, Inc., Beverly, MA) and total forms of ERK1/2, STAT1, and STAT3, LIFR (Santa Cruz, Santa Cruz, CA).

    Techniques: