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  • 99
    Name:
    Epidermal Growth Factor Human EGF
    Description:
    The human EGF coding cDNA was obtained from human periodontal tissue mRNA subcloned into a prokaryotic expression vector and expressed in E coli Epidermal Growth Factor Human EGF was purified and stored in PBS buffer containing 0 1 BSA
    Catalog Number:
    9908
    Price:
    None
    Category:
    Cytokines
    Source:
    Human Recombinant Protein
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc total egfr
    Elucidation of mechanisms of AC0010 resistance by RNAseq profiling. Total RNA was isolated and subjected to RNAseq profiling as detailed in the Materials and Methods section. Shown are genes in p53 and TGFβ pathways enriched in H1975-P1 cells (A), and apoptosis and NFκB pathways in H1975-AVR1 cells (B). Real-time PCR analysis to confirm increased expression of <t>c-MET</t> and BCL-2 in H1975-P1-R1 and H1975-AVR1-R2 cells, respectively (C). Western blot analysis of the levels of c-MET and BCL-2 in H1975-P1-R1 and H1975-AVR1-R2 cells as well as the phosphorylation status of c-MET, <t>EGFR,</t> and their downstream molecules (AKT1 and ERK) (D).
    The human EGF coding cDNA was obtained from human periodontal tissue mRNA subcloned into a prokaryotic expression vector and expressed in E coli Epidermal Growth Factor Human EGF was purified and stored in PBS buffer containing 0 1 BSA
    https://www.bioz.com/result/total egfr/product/Cell Signaling Technology Inc
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    total egfr - by Bioz Stars, 2021-05
    99/100 stars

    Images

    1) Product Images from "Overcoming Resistance to AC0010, a Third Generation of EGFR Inhibitor, by Targeting c-MET and BCL-2"

    Article Title: Overcoming Resistance to AC0010, a Third Generation of EGFR Inhibitor, by Targeting c-MET and BCL-2

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2018.11.004

    Elucidation of mechanisms of AC0010 resistance by RNAseq profiling. Total RNA was isolated and subjected to RNAseq profiling as detailed in the Materials and Methods section. Shown are genes in p53 and TGFβ pathways enriched in H1975-P1 cells (A), and apoptosis and NFκB pathways in H1975-AVR1 cells (B). Real-time PCR analysis to confirm increased expression of c-MET and BCL-2 in H1975-P1-R1 and H1975-AVR1-R2 cells, respectively (C). Western blot analysis of the levels of c-MET and BCL-2 in H1975-P1-R1 and H1975-AVR1-R2 cells as well as the phosphorylation status of c-MET, EGFR, and their downstream molecules (AKT1 and ERK) (D).
    Figure Legend Snippet: Elucidation of mechanisms of AC0010 resistance by RNAseq profiling. Total RNA was isolated and subjected to RNAseq profiling as detailed in the Materials and Methods section. Shown are genes in p53 and TGFβ pathways enriched in H1975-P1 cells (A), and apoptosis and NFκB pathways in H1975-AVR1 cells (B). Real-time PCR analysis to confirm increased expression of c-MET and BCL-2 in H1975-P1-R1 and H1975-AVR1-R2 cells, respectively (C). Western blot analysis of the levels of c-MET and BCL-2 in H1975-P1-R1 and H1975-AVR1-R2 cells as well as the phosphorylation status of c-MET, EGFR, and their downstream molecules (AKT1 and ERK) (D).

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Expressing, Western Blot

    Overcoming AC0010 resistance by targeting c-MET in cell culture model. (A) H1975-P1-R1 cells were transfected with siRNA targeting c-MET, along with the control siRNA, and the sensitivity of transfected cells to AC0010 was determined by WST-1 assay. Western blot analysis was performed to show c-MET knockdown. (B) Growth curve of H1975-P1-R1 cells treated with various concentrations of crizotinib. (C) Growth curve of H1975-P1-R1 cells treated with various concentrations of AC0010 in combination with 2 μM crizotinib or 0.5 μM crizotinib ( n = 3). (D) Growth curve of H1975-P1-R1 cells treated with 1 μM AC0010 in combination with various concentrations of crizotinib ( n = 3). (E) Clonogenic survival assays of H1975-P1-R1 cells treated with AC0010, criztonib, or the combination with indicated concentrations ( n = 3). (F) Western blot analysis of the phosphorylation status of c-MET, EGFR, and their downstream molecules (AKT1 and ERK) in H1975-P1-R1 cells treated with indicated drugs and concentrations.
    Figure Legend Snippet: Overcoming AC0010 resistance by targeting c-MET in cell culture model. (A) H1975-P1-R1 cells were transfected with siRNA targeting c-MET, along with the control siRNA, and the sensitivity of transfected cells to AC0010 was determined by WST-1 assay. Western blot analysis was performed to show c-MET knockdown. (B) Growth curve of H1975-P1-R1 cells treated with various concentrations of crizotinib. (C) Growth curve of H1975-P1-R1 cells treated with various concentrations of AC0010 in combination with 2 μM crizotinib or 0.5 μM crizotinib ( n = 3). (D) Growth curve of H1975-P1-R1 cells treated with 1 μM AC0010 in combination with various concentrations of crizotinib ( n = 3). (E) Clonogenic survival assays of H1975-P1-R1 cells treated with AC0010, criztonib, or the combination with indicated concentrations ( n = 3). (F) Western blot analysis of the phosphorylation status of c-MET, EGFR, and their downstream molecules (AKT1 and ERK) in H1975-P1-R1 cells treated with indicated drugs and concentrations.

    Techniques Used: Cell Culture, Transfection, WST-1 Assay, Western Blot

    2) Product Images from "Anti-inflammatory effect of afatinib (an EGFR-TKI) on OGD-induced neuroinflammation"

    Article Title: Anti-inflammatory effect of afatinib (an EGFR-TKI) on OGD-induced neuroinflammation

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-38676-7

    Afatinib blocked OGD-induced EGFR activation and downstream signalings. ( A , B ) CTX-TNA2 cells were exposed to oxygen-glucose deprivation (OGD) for various durations (3, 6, 12 h). Western blot assay was employed to measure ( A ) the levels of phospho-EGFR (pEGFR) and total-EGFR (EGFR) as well as ( B ) phospho-ERK (pERK), total-ERK (ERK), phospho-AKT (pAKT), total-AKT (AKT) and β-Actin. Each lane contained 40 μg protein for all experiments. Graphs show statistical results of pEGFR and EGFR ( A ) as well as pERK, ERK, pAKT and AKT ( B ) from relative optical density of bands on the blots. *P
    Figure Legend Snippet: Afatinib blocked OGD-induced EGFR activation and downstream signalings. ( A , B ) CTX-TNA2 cells were exposed to oxygen-glucose deprivation (OGD) for various durations (3, 6, 12 h). Western blot assay was employed to measure ( A ) the levels of phospho-EGFR (pEGFR) and total-EGFR (EGFR) as well as ( B ) phospho-ERK (pERK), total-ERK (ERK), phospho-AKT (pAKT), total-AKT (AKT) and β-Actin. Each lane contained 40 μg protein for all experiments. Graphs show statistical results of pEGFR and EGFR ( A ) as well as pERK, ERK, pAKT and AKT ( B ) from relative optical density of bands on the blots. *P

    Techniques Used: Activation Assay, Western Blot

    3) Product Images from "Synergistic Effect of MiR-146a Mimic and Cetuximab on Hepatocellular Carcinoma Cells"

    Article Title: Synergistic Effect of MiR-146a Mimic and Cetuximab on Hepatocellular Carcinoma Cells

    Journal: BioMed Research International

    doi: 10.1155/2014/384121

    Effect of combining miR-146a mimic and cetuximab on EGFR and its downstream pathway signals in HCC HepG2 cells. HepG2 cells (2.5 × 10 4 cells per well in 24-well plate) were cultured for 24 h then transfected with miR-146a mimic and negative control (200 nM). Meanwhile, cetuximab was added and the cells were cultured up to another 96 h. Western blot and signal intensity of the bands were shown. Antibodies included phospho-EGFR (p-EGFR), total-EGFR (t-EGFR), p-ERK1/2, t-ERK1/2, p-AKT, t-AKT, p-stat5, t-stat5, and β -Actin. M: mock control; Cetu: cetuximab; Mimi: miR-146a mimic; comb: combination of cetuximab and miR-146a mimic.
    Figure Legend Snippet: Effect of combining miR-146a mimic and cetuximab on EGFR and its downstream pathway signals in HCC HepG2 cells. HepG2 cells (2.5 × 10 4 cells per well in 24-well plate) were cultured for 24 h then transfected with miR-146a mimic and negative control (200 nM). Meanwhile, cetuximab was added and the cells were cultured up to another 96 h. Western blot and signal intensity of the bands were shown. Antibodies included phospho-EGFR (p-EGFR), total-EGFR (t-EGFR), p-ERK1/2, t-ERK1/2, p-AKT, t-AKT, p-stat5, t-stat5, and β -Actin. M: mock control; Cetu: cetuximab; Mimi: miR-146a mimic; comb: combination of cetuximab and miR-146a mimic.

    Techniques Used: Cell Culture, Transfection, Negative Control, Western Blot

    Effect of miR-146a on EGFR and its downstream pathway signals in HCC HepG2 cells. HepG2 cells (2.5 × 10 4 cells per well in 24-well plate) were cultured for 24 h then transfected with miR-146a inhibitor, miR-146a mimic, EGFR siRNA, and their negative controls (200 nM) up to another 96 h. Western blot and signal intensity of the bands were shown. Antibodies included phospho-EGFR (p-EGFR), total-EGFR (t-EGFR), p-ERK1/2, t-ERK1/2, p-AKT, t-AKT, p-stat5, t-stat5, and β -Actin. M: mock control; C1: negative control for miRNA inhibitor; Inhi: miR-146a inhibitor; C2: negative control for miRNA mimic; Mimi: miR-146a mimic; Si: EGFR siRNA.
    Figure Legend Snippet: Effect of miR-146a on EGFR and its downstream pathway signals in HCC HepG2 cells. HepG2 cells (2.5 × 10 4 cells per well in 24-well plate) were cultured for 24 h then transfected with miR-146a inhibitor, miR-146a mimic, EGFR siRNA, and their negative controls (200 nM) up to another 96 h. Western blot and signal intensity of the bands were shown. Antibodies included phospho-EGFR (p-EGFR), total-EGFR (t-EGFR), p-ERK1/2, t-ERK1/2, p-AKT, t-AKT, p-stat5, t-stat5, and β -Actin. M: mock control; C1: negative control for miRNA inhibitor; Inhi: miR-146a inhibitor; C2: negative control for miRNA mimic; Mimi: miR-146a mimic; Si: EGFR siRNA.

    Techniques Used: Cell Culture, Transfection, Western Blot, Negative Control

    4) Product Images from "Shikonin causes cell-cycle arrest and induces apoptosis by regulating the EGFR–NF-κB signalling pathway in human epidermoid carcinoma A431 cells"

    Article Title: Shikonin causes cell-cycle arrest and induces apoptosis by regulating the EGFR–NF-κB signalling pathway in human epidermoid carcinoma A431 cells

    Journal: Bioscience Reports

    doi: 10.1042/BSR20150002

    Shikonin inactivated NF-κB via inhibiting EGFR signalling pathways in A431 cell The A431 cells were treated with various concentrations of shikonin (0, 2.5, 5 and 10 μM) for 2 h. ( A ) Western blot analysis was performed for p-EGFR and t-EGFR, p-STAT3 and t-STAT3, p-Akt and t-Akt respectively. The A431 cells were pre-treated with various concentrations of shikonin (0, 2.5, 5 and 10 μM) for 2 h, followed by treatment with EGF (100 ng/ml) for 10 min. The EGFR inhibitor (AG1478, 20 μM), Stattic (STAT3 inhibitor, 2 μM) and LY294002 (PI3K inhibitor, 10 μM) were used as a positive drug control respectively. ( B ) Protein expression levels of p-EGFR and t-EGFR, p-STAT3 and t-STAT3, p-Akt and t-Akt were detected by western blot analysis. A431 cells were pre-treated with or without AG1478 for 2 h and then were treated with shikonin (10 μM) for 2 h. ( C ) NF-κB DNA-binding activity assay. All data are presented as mean ± S.E.M., n =6. * P
    Figure Legend Snippet: Shikonin inactivated NF-κB via inhibiting EGFR signalling pathways in A431 cell The A431 cells were treated with various concentrations of shikonin (0, 2.5, 5 and 10 μM) for 2 h. ( A ) Western blot analysis was performed for p-EGFR and t-EGFR, p-STAT3 and t-STAT3, p-Akt and t-Akt respectively. The A431 cells were pre-treated with various concentrations of shikonin (0, 2.5, 5 and 10 μM) for 2 h, followed by treatment with EGF (100 ng/ml) for 10 min. The EGFR inhibitor (AG1478, 20 μM), Stattic (STAT3 inhibitor, 2 μM) and LY294002 (PI3K inhibitor, 10 μM) were used as a positive drug control respectively. ( B ) Protein expression levels of p-EGFR and t-EGFR, p-STAT3 and t-STAT3, p-Akt and t-Akt were detected by western blot analysis. A431 cells were pre-treated with or without AG1478 for 2 h and then were treated with shikonin (10 μM) for 2 h. ( C ) NF-κB DNA-binding activity assay. All data are presented as mean ± S.E.M., n =6. * P

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

    5) Product Images from "SOX2 expression is an early event in a murine model of EGFR mutant lung cancer and promotes proliferation of a subset of EGFR mutant lung adenocarcinoma cell lines"

    Article Title: SOX2 expression is an early event in a murine model of EGFR mutant lung cancer and promotes proliferation of a subset of EGFR mutant lung adenocarcinoma cell lines

    Journal: Lung cancer (Amsterdam, Netherlands)

    doi: 10.1016/j.lungcan.2014.03.021

    Elevated EGFR signaling correlates with SOX2 expression in lung tissues in vivo . A) Immunoblots of whole lung lysates of CCSP-rtTA/TetO- EGFR L858R+T790M mutant mice treated with DOX for 3 and 9 weeks. B) Immunohistochemical staining of CCSP-rtTA/TetO-egfr
    Figure Legend Snippet: Elevated EGFR signaling correlates with SOX2 expression in lung tissues in vivo . A) Immunoblots of whole lung lysates of CCSP-rtTA/TetO- EGFR L858R+T790M mutant mice treated with DOX for 3 and 9 weeks. B) Immunohistochemical staining of CCSP-rtTA/TetO-egfr

    Techniques Used: Expressing, In Vivo, Western Blot, Mutagenesis, Mouse Assay, Immunohistochemistry, Staining

    A) Erlotinib alters the expression of SOX2 and OCT4 in EGFR mutant NSCLC cell lines. Immunoblot analysis of H1975 and HCC827 cells treated with varying doses of erlotinib for 3 and 9 days. C=vehicle control. B) PI3K/AKT pathway inhibitors decrease SOX2
    Figure Legend Snippet: A) Erlotinib alters the expression of SOX2 and OCT4 in EGFR mutant NSCLC cell lines. Immunoblot analysis of H1975 and HCC827 cells treated with varying doses of erlotinib for 3 and 9 days. C=vehicle control. B) PI3K/AKT pathway inhibitors decrease SOX2

    Techniques Used: Expressing, Mutagenesis

    6) Product Images from "Identification of the angiogenic gene signature induced by EGF and hypoxia in colorectal cancer"

    Article Title: Identification of the angiogenic gene signature induced by EGF and hypoxia in colorectal cancer

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-13-518

    EGF receptor is autophosphorylated in Caco-2 and activates downstream signalling pathways. Caco-2 cells were stimulated with 20 ng/mL EGF for the time periods indicated. Western blotting for (a) phosphorylated EGFR or total EGFR and (b) antibodies recognising signalling enzymes is shown. Cell lysate of EGF-treated A431 cells was used as positive control. α-tubulin is shown as a loading control. Densitometry was performed using Phoretix 1D analysis software against α-tubulin (for ERK, data for p42 and p44 are shown).
    Figure Legend Snippet: EGF receptor is autophosphorylated in Caco-2 and activates downstream signalling pathways. Caco-2 cells were stimulated with 20 ng/mL EGF for the time periods indicated. Western blotting for (a) phosphorylated EGFR or total EGFR and (b) antibodies recognising signalling enzymes is shown. Cell lysate of EGF-treated A431 cells was used as positive control. α-tubulin is shown as a loading control. Densitometry was performed using Phoretix 1D analysis software against α-tubulin (for ERK, data for p42 and p44 are shown).

    Techniques Used: Western Blot, Positive Control, Software

    7) Product Images from "Uropathogenic Escherichia coli invades bladder epithelial cells by activating kinase networks in host cells"

    Article Title: Uropathogenic Escherichia coli invades bladder epithelial cells by activating kinase networks in host cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003499

    Role of EGFR, Akt, and mTORCs in UTI89 invasion of mouse bladder. A and B , strips of mouse transitional epithelial tissue were individually placed in wells (12-well plates) and treated or not with the indicated inhibitor for 1 h at 37 °C. UPEC (5 × 10 7 /ml) were added for additional 2 h, and tissue strips were washed three times with PBS. The tissue strips were lysed in PBS containing 0.1% (v/v) Triton X-100. For one part ( A ), cell lysates were analyzed for EGFR and Akt phosphorylation by Western blotting using the indicated antibodies. GAPDH was used as a protein loading control. For the second part ( B ), dilutions of lysates were plated on LB agar plates to allow colony growth. Colonies were counted, and data are presented as the -fold change of control values. Statistical analysis was performed by Student's t test compared with the nontreated ( NT ) control. *, p
    Figure Legend Snippet: Role of EGFR, Akt, and mTORCs in UTI89 invasion of mouse bladder. A and B , strips of mouse transitional epithelial tissue were individually placed in wells (12-well plates) and treated or not with the indicated inhibitor for 1 h at 37 °C. UPEC (5 × 10 7 /ml) were added for additional 2 h, and tissue strips were washed three times with PBS. The tissue strips were lysed in PBS containing 0.1% (v/v) Triton X-100. For one part ( A ), cell lysates were analyzed for EGFR and Akt phosphorylation by Western blotting using the indicated antibodies. GAPDH was used as a protein loading control. For the second part ( B ), dilutions of lysates were plated on LB agar plates to allow colony growth. Colonies were counted, and data are presented as the -fold change of control values. Statistical analysis was performed by Student's t test compared with the nontreated ( NT ) control. *, p

    Techniques Used: Western Blot

    8) Product Images from "A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity"

    Article Title: A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity

    Journal: mAbs

    doi: 10.4161/mabs.3.3.15188

    Binding activities of EI-04 BsAb and its parental anti-EGFR and anti-IGF-1R mAbs. (A and B) Biacore surface plasmon resonance sensorgrams of soluble EGFR ectodomain (0–30 nM) to chip-captured EI-04 (A) and anti-EGFR mAb M60-A02 (B). (C) Octet
    Figure Legend Snippet: Binding activities of EI-04 BsAb and its parental anti-EGFR and anti-IGF-1R mAbs. (A and B) Biacore surface plasmon resonance sensorgrams of soluble EGFR ectodomain (0–30 nM) to chip-captured EI-04 (A) and anti-EGFR mAb M60-A02 (B). (C) Octet

    Techniques Used: Binding Assay, SPR Assay, Chromatin Immunoprecipitation

    Dual inhibitory activities of EI-04 on EGFR and IGF-1R. (A) Blockade of europium-labeled EGF binding to EGFR-Fc by EI-04 in comparison to various anti-EGFR mAbs. (B) Blockade of a constant level of IGF-1 (25 nM, left part) or IGF-2 (60 nM, right part)
    Figure Legend Snippet: Dual inhibitory activities of EI-04 on EGFR and IGF-1R. (A) Blockade of europium-labeled EGF binding to EGFR-Fc by EI-04 in comparison to various anti-EGFR mAbs. (B) Blockade of a constant level of IGF-1 (25 nM, left part) or IGF-2 (60 nM, right part)

    Techniques Used: Labeling, Binding Assay

    Concurrent blockade of EGFR and IGF-1R signaling pathways and enhanced inhibition of tumor cell growth and cell cycle progression by EI-04. (A) Simultaneous inhibition of phosphorylation of EGFR and IGF-1R in HN11 tumor cells by EI-04. Cells grown in
    Figure Legend Snippet: Concurrent blockade of EGFR and IGF-1R signaling pathways and enhanced inhibition of tumor cell growth and cell cycle progression by EI-04. (A) Simultaneous inhibition of phosphorylation of EGFR and IGF-1R in HN11 tumor cells by EI-04. Cells grown in

    Techniques Used: Inhibition

    Schematic diagram and biochemical analysis of EI-04. (A) Schematic diagram of EI-04, composed of an anti-EGFR Fab linked to an effectorless human Fc with a stability-engineered anti-IGF-1R scFv attached at the C-terminus. (B) SDS-PAGE analysis of EI-04
    Figure Legend Snippet: Schematic diagram and biochemical analysis of EI-04. (A) Schematic diagram of EI-04, composed of an anti-EGFR Fab linked to an effectorless human Fc with a stability-engineered anti-IGF-1R scFv attached at the C-terminus. (B) SDS-PAGE analysis of EI-04

    Techniques Used: SDS Page

    9) Product Images from "Dual targeting of HER3 and MEK may overcome HER3-dependent drug-resistance of colon cancers"

    Article Title: Dual targeting of HER3 and MEK may overcome HER3-dependent drug-resistance of colon cancers

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11400

    Patritumab administration inhibits the PI3K survival pathway and activates the MAPK proliferation pathway Total cell lysates from HT29 ( A ), Pt-1 ( B ), Pt-2 ( C ), and Pt-3 ( D ) cells, incubated with 10 ng/ml HRG-β1 and 10 μg/ml patritumab antibody for the indicated times, were analyzed by immunoblot to evaluate the expression of total and P-HER3, HER2, EGFR, total and p-AKT, total and p-ERK. The anti-HSP70 antibody was used to validate equivalent amount of loaded proteins in each lane.
    Figure Legend Snippet: Patritumab administration inhibits the PI3K survival pathway and activates the MAPK proliferation pathway Total cell lysates from HT29 ( A ), Pt-1 ( B ), Pt-2 ( C ), and Pt-3 ( D ) cells, incubated with 10 ng/ml HRG-β1 and 10 μg/ml patritumab antibody for the indicated times, were analyzed by immunoblot to evaluate the expression of total and P-HER3, HER2, EGFR, total and p-AKT, total and p-ERK. The anti-HSP70 antibody was used to validate equivalent amount of loaded proteins in each lane.

    Techniques Used: Incubation, Expressing

    10) Product Images from "Overcoming EGFRG724S-mediated osimertinib resistance through unique binding characteristics of second-generation EGFR inhibitors"

    Article Title: Overcoming EGFRG724S-mediated osimertinib resistance through unique binding characteristics of second-generation EGFR inhibitors

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07078-0

    Kinetic evaluation of second- and third-generation EGFR TKIs against EGFR mutant proteins. a Schematic overview of two-step binding mechanism of covalent inhibitors to kinases with K i (quotient of k off and k on ) describing the reversible binding affinity and k inact describing the rate of inactivation. b Time-dependent IC 50 -determination of afatinib and osimertinib on EGFR mutant proteins. Representative curves of single measurements in duplicates are shown. c Heatmap of biochemical IC 50 -, K i -, and k inact determination of second- and third-generation EGFR TKIs against EGFR mutant proteins. Values are the mean of three independent measurements in duplicates. d Immunoblotting results of NIH-3T3 cells (EGFR 19del or EGFR 19del+G724S ) monitoring phospho-EGFR and total EGFR. Cells were treated for indicated times (0, 1, 3, 6, and 24 h) with osimertinib (1 µM) or afatinib (1 µM). HSP90 was used as loading control ( n = 3)
    Figure Legend Snippet: Kinetic evaluation of second- and third-generation EGFR TKIs against EGFR mutant proteins. a Schematic overview of two-step binding mechanism of covalent inhibitors to kinases with K i (quotient of k off and k on ) describing the reversible binding affinity and k inact describing the rate of inactivation. b Time-dependent IC 50 -determination of afatinib and osimertinib on EGFR mutant proteins. Representative curves of single measurements in duplicates are shown. c Heatmap of biochemical IC 50 -, K i -, and k inact determination of second- and third-generation EGFR TKIs against EGFR mutant proteins. Values are the mean of three independent measurements in duplicates. d Immunoblotting results of NIH-3T3 cells (EGFR 19del or EGFR 19del+G724S ) monitoring phospho-EGFR and total EGFR. Cells were treated for indicated times (0, 1, 3, 6, and 24 h) with osimertinib (1 µM) or afatinib (1 µM). HSP90 was used as loading control ( n = 3)

    Techniques Used: Mutagenesis, Binding Assay

    EGFR G724S mediates resistance to third-generation EGFR inhibitors in vitro and in vivo. a Immunoblotting results of NIH-3T3 cells (empty vector, EGFR 19del , EGFR G724S , or EGFR 19del+G724S ) monitoring phospho-EGFR and total EGFR under erlotinib treatment (24 h). HSP90 was used as loading control. b Immunoblotting of NIH-3T3 cells under osimertinib treatment (24 h) is shown. Dose–response measurement of Ba/F3 cells expressing EGFR 19del , EGFR G724S , or EGFR 19del+G724S treated for 72 h with c erlotinib or d osimertinib. Experiments were performed at least three times. e , f Graphs show tumor volume of mice injected with NIH-3T3 EGFR 19del and EGFR 19del+G724S cells treated with osimertinib (5 mg/kg, i.p., once daily). e Tumor volumes ( EGFR 19del vehicle, n = 7 mice; EGFR 19del osimertinib, n = 8 mice; EGFR 19del+G724S vehicle, n = 7 mice; EGFR 19del+G724S osimertinib, n =10 mice) were assessed for 20 days by longitudinal caliper measurements every second day following treatment initiation. f Tumor volumes were quantified after 8 days of treatment. Volume changes in the osimertinib treatment cohort (dark gray and green) were compared with the vehicle-treated control group (light gray and green). Each dot represents a single tumor per mouse. Significance is calculated by two-tailed Student’s t test, n.s.: non-significant. g Representative images of Cleaved Caspase-3 stainings. Tumors of mice bearing NIH-3T3 EGFR 19del or EGFR 19del+G724S cells were treated with vehicle solution HPMC (0, 5%) or osimertinib. Scale bar 100 μm. h Quantification of Cleaved Caspase-3 staining. Number of apoptotic cells in the osimertinib-treated cohort (dark gray and green) was compared with the vehicle-treated control group (light gray and green)
    Figure Legend Snippet: EGFR G724S mediates resistance to third-generation EGFR inhibitors in vitro and in vivo. a Immunoblotting results of NIH-3T3 cells (empty vector, EGFR 19del , EGFR G724S , or EGFR 19del+G724S ) monitoring phospho-EGFR and total EGFR under erlotinib treatment (24 h). HSP90 was used as loading control. b Immunoblotting of NIH-3T3 cells under osimertinib treatment (24 h) is shown. Dose–response measurement of Ba/F3 cells expressing EGFR 19del , EGFR G724S , or EGFR 19del+G724S treated for 72 h with c erlotinib or d osimertinib. Experiments were performed at least three times. e , f Graphs show tumor volume of mice injected with NIH-3T3 EGFR 19del and EGFR 19del+G724S cells treated with osimertinib (5 mg/kg, i.p., once daily). e Tumor volumes ( EGFR 19del vehicle, n = 7 mice; EGFR 19del osimertinib, n = 8 mice; EGFR 19del+G724S vehicle, n = 7 mice; EGFR 19del+G724S osimertinib, n =10 mice) were assessed for 20 days by longitudinal caliper measurements every second day following treatment initiation. f Tumor volumes were quantified after 8 days of treatment. Volume changes in the osimertinib treatment cohort (dark gray and green) were compared with the vehicle-treated control group (light gray and green). Each dot represents a single tumor per mouse. Significance is calculated by two-tailed Student’s t test, n.s.: non-significant. g Representative images of Cleaved Caspase-3 stainings. Tumors of mice bearing NIH-3T3 EGFR 19del or EGFR 19del+G724S cells were treated with vehicle solution HPMC (0, 5%) or osimertinib. Scale bar 100 μm. h Quantification of Cleaved Caspase-3 staining. Number of apoptotic cells in the osimertinib-treated cohort (dark gray and green) was compared with the vehicle-treated control group (light gray and green)

    Techniques Used: In Vitro, In Vivo, Plasmid Preparation, Expressing, Mouse Assay, Injection, Two Tailed Test, Staining

    Biochemical profiling of EGFR G724S . a Homogeneous time-resolved fluorescence (HTRF) is used for IC 50 -determination for EGFR 19del and EGFR 19del +G724S with representative inhibitors. Representative dose–response curves of a single measurement in duplicates are shown. b Comparison of biochemical IC 50 -values with HTRF for the three generations of EGFR TKIs against EGFR 19del+G724S . Values are the mean of three independent measurements in duplicates. c Immunoblotting results of NIH-3T3 cells (empty vector, EGFR 19del , EGFR G724S or EGFR 19del+G724S ) showing phospho-EGFR and total EGFR under afatinib treatment (24 h). HSP90 was used as loading control ( n = 3). d Dose–response measurement of Ba/F3 cells expressing EGFR 19del , EGFR G724S , or EGFR 19del+G724S treated for 72 h with afatinib. Experiments were accomplished for at least three times. e Structure of exon 20 mutant EGFR , bound to 4-anilinoquinazoline based TKI PD168393, is shown
    Figure Legend Snippet: Biochemical profiling of EGFR G724S . a Homogeneous time-resolved fluorescence (HTRF) is used for IC 50 -determination for EGFR 19del and EGFR 19del +G724S with representative inhibitors. Representative dose–response curves of a single measurement in duplicates are shown. b Comparison of biochemical IC 50 -values with HTRF for the three generations of EGFR TKIs against EGFR 19del+G724S . Values are the mean of three independent measurements in duplicates. c Immunoblotting results of NIH-3T3 cells (empty vector, EGFR 19del , EGFR G724S or EGFR 19del+G724S ) showing phospho-EGFR and total EGFR under afatinib treatment (24 h). HSP90 was used as loading control ( n = 3). d Dose–response measurement of Ba/F3 cells expressing EGFR 19del , EGFR G724S , or EGFR 19del+G724S treated for 72 h with afatinib. Experiments were accomplished for at least three times. e Structure of exon 20 mutant EGFR , bound to 4-anilinoquinazoline based TKI PD168393, is shown

    Techniques Used: Fluorescence, Plasmid Preparation, Expressing, Mutagenesis

    11) Product Images from "Influence of Matrices on 3D-Cultured Prostate Cancer Cells' Drug Response and Expression of Drug-Action Associated Proteins"

    Article Title: Influence of Matrices on 3D-Cultured Prostate Cancer Cells' Drug Response and Expression of Drug-Action Associated Proteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158116

    Total EGFR and β-III tubulin protein expression in DU145 cells. A representative image of total p53 protein expression in 2D- and 3D-cultured LNCaP cells. Bars represent the relative protein quantification of p53 isoforms at ~144, ~100, ~58, and ~44 kda on the basis of β-actin, and indicate the mean ± SEM (n = 3 for each group). *p≤0.05 using one-way ANOVA and unpaired t-tests.
    Figure Legend Snippet: Total EGFR and β-III tubulin protein expression in DU145 cells. A representative image of total p53 protein expression in 2D- and 3D-cultured LNCaP cells. Bars represent the relative protein quantification of p53 isoforms at ~144, ~100, ~58, and ~44 kda on the basis of β-actin, and indicate the mean ± SEM (n = 3 for each group). *p≤0.05 using one-way ANOVA and unpaired t-tests.

    Techniques Used: Expressing, Cell Culture

    Total EGFR and β-III tubulin protein expression in DU145 cells. A representative image of the expression of (A) total EGFR and (B) β-III tubulin in 2D- and 3D-cultured DU145 cells. Bars represent the relative protein quantification of (A) total EGFR and (B) β-III tubulin on the basis of GAPDH and β-actin, and indicate the mean ± SEM (n = 3 for each group). *p≤0.05, **p≤0.01, and ***p≤0.001, using one-way ANOVA and unpaired t-tests.
    Figure Legend Snippet: Total EGFR and β-III tubulin protein expression in DU145 cells. A representative image of the expression of (A) total EGFR and (B) β-III tubulin in 2D- and 3D-cultured DU145 cells. Bars represent the relative protein quantification of (A) total EGFR and (B) β-III tubulin on the basis of GAPDH and β-actin, and indicate the mean ± SEM (n = 3 for each group). *p≤0.05, **p≤0.01, and ***p≤0.001, using one-way ANOVA and unpaired t-tests.

    Techniques Used: Expressing, Cell Culture

    12) Product Images from "Elevated Expression of Fn14 in Non-Small Cell Lung Cancer Correlates with Activated EGFR and Promotes Tumor Cell Migration and Invasion"

    Article Title: Elevated Expression of Fn14 in Non-Small Cell Lung Cancer Correlates with Activated EGFR and Promotes Tumor Cell Migration and Invasion

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2012.03.026

    Erlotinib treatment of TKI-sensitive NSCLC cells decreases Fn14 expression. A: Serum-starved HCC827 cells containing the EGFR E746-A750–activating mutation were treated with vehicle for 12 hours or 1 μmol/L erlotinib for the indicated
    Figure Legend Snippet: Erlotinib treatment of TKI-sensitive NSCLC cells decreases Fn14 expression. A: Serum-starved HCC827 cells containing the EGFR E746-A750–activating mutation were treated with vehicle for 12 hours or 1 μmol/L erlotinib for the indicated

    Techniques Used: Expressing, Mutagenesis

    13) Product Images from "Fibrinogen inhibits neurite outgrowth via ?3 integrin-mediated phosphorylation of the EGF receptor"

    Article Title: Fibrinogen inhibits neurite outgrowth via ?3 integrin-mediated phosphorylation of the EGF receptor

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0704045104

    Proposed model of fibrinogen-mediated inhibition of neurite outgrowth. Traumatic injury or other neurodegenerative conditions associated with compromised BBB or vascular damage allow the leakage of fibrinogen in the CNS. Fibrinogen binding to β3 integrin on neurons induces coclustering of β3 integrin with EGFR, leading to EGFR phosphorylation and subsequent inhibition of neurite outgrowth.
    Figure Legend Snippet: Proposed model of fibrinogen-mediated inhibition of neurite outgrowth. Traumatic injury or other neurodegenerative conditions associated with compromised BBB or vascular damage allow the leakage of fibrinogen in the CNS. Fibrinogen binding to β3 integrin on neurons induces coclustering of β3 integrin with EGFR, leading to EGFR phosphorylation and subsequent inhibition of neurite outgrowth.

    Techniques Used: Inhibition, Binding Assay

    Fibrinogen induces transactivation of EGFR via β3 integrin to inhibit neurite outgrowth. ( A and B ) Treatment with the EGFR phosphorylation inhibitor PD168393 (50 nM) on 1.5 mg/ml fibrinogen treatment results in a statistically significant increase in the percentage of mouse CGNs bearing neurites ( A ; P
    Figure Legend Snippet: Fibrinogen induces transactivation of EGFR via β3 integrin to inhibit neurite outgrowth. ( A and B ) Treatment with the EGFR phosphorylation inhibitor PD168393 (50 nM) on 1.5 mg/ml fibrinogen treatment results in a statistically significant increase in the percentage of mouse CGNs bearing neurites ( A ; P

    Techniques Used:

    14) Product Images from "Estrogen promotes the brain metastatic colonization of triple negative breast cancer cells via an astrocyte-mediated paracrine mechanism"

    Article Title: Estrogen promotes the brain metastatic colonization of triple negative breast cancer cells via an astrocyte-mediated paracrine mechanism

    Journal: Oncogene

    doi: 10.1038/onc.2015.353

    A simplified scheme of the paracrine effects of estrogen in brain metastases Consistent with the notion that breast cancer cells benefit from normal homeostasis mechanisms at the metastatic site ( 48 , 50 ), our data supports a novel mechanism whereby brain metastatic cells exploit estrogen signaling though ER+ astrocytes. Estrogen can activate ERs on astrocytes, leading to transcriptional upregulation of EGFR ligands, activation of EGFR on TN EGFR+ brain metastatic breast cancer cells, and upregulation of several metastatic mediators, including S100A4. Given the complex bi-directional interactions between astrocytes and breast cancer cells, it is possible that other factors regulated by ERs in astrocytes and receptors on brain metastatic cells participate in this process. Ovarian and local estrogen depletion decrease brain metastatic colonization of TN EGFR+ 231BR cells in vivo , emphasizing the need to consider the brain microenvironment in designing novel therapies for brain metastases and the possibility to extend the use of aromatase inhibitors in the prevention of brain metastases in TN EGFR+ patients.
    Figure Legend Snippet: A simplified scheme of the paracrine effects of estrogen in brain metastases Consistent with the notion that breast cancer cells benefit from normal homeostasis mechanisms at the metastatic site ( 48 , 50 ), our data supports a novel mechanism whereby brain metastatic cells exploit estrogen signaling though ER+ astrocytes. Estrogen can activate ERs on astrocytes, leading to transcriptional upregulation of EGFR ligands, activation of EGFR on TN EGFR+ brain metastatic breast cancer cells, and upregulation of several metastatic mediators, including S100A4. Given the complex bi-directional interactions between astrocytes and breast cancer cells, it is possible that other factors regulated by ERs in astrocytes and receptors on brain metastatic cells participate in this process. Ovarian and local estrogen depletion decrease brain metastatic colonization of TN EGFR+ 231BR cells in vivo , emphasizing the need to consider the brain microenvironment in designing novel therapies for brain metastases and the possibility to extend the use of aromatase inhibitors in the prevention of brain metastases in TN EGFR+ patients.

    Techniques Used: Activation Assay, In Vivo

    E2 upregulates EGFR ligands in astrocytes and results in EGFR activation in brain metastatic cells a. Primary mouse astrocytes were treated with vehicle (OH) or 10 nM E2 for the indicated times and qRT-PCR was used to measure Egf, Tgfα and Ereg mRNA levels. Bars represent fold change mRNA levels normalized to mouse β-Actin mRNA and relative to OH-treated. *P
    Figure Legend Snippet: E2 upregulates EGFR ligands in astrocytes and results in EGFR activation in brain metastatic cells a. Primary mouse astrocytes were treated with vehicle (OH) or 10 nM E2 for the indicated times and qRT-PCR was used to measure Egf, Tgfα and Ereg mRNA levels. Bars represent fold change mRNA levels normalized to mouse β-Actin mRNA and relative to OH-treated. *P

    Techniques Used: Activation Assay, Quantitative RT-PCR

    15) Product Images from "Overcoming EGFRG724S-mediated osimertinib resistance through unique binding characteristics of second-generation EGFR inhibitors"

    Article Title: Overcoming EGFRG724S-mediated osimertinib resistance through unique binding characteristics of second-generation EGFR inhibitors

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07078-0

    Kinetic evaluation of second- and third-generation EGFR TKIs against EGFR mutant proteins. a Schematic overview of two-step binding mechanism of covalent inhibitors to kinases with K i (quotient of k off and k on ) describing the reversible binding affinity and k inact describing the rate of inactivation. b Time-dependent IC 50 -determination of afatinib and osimertinib on EGFR mutant proteins. Representative curves of single measurements in duplicates are shown. c Heatmap of biochemical IC 50 -, K i -, and k inact determination of second- and third-generation EGFR TKIs against EGFR mutant proteins. Values are the mean of three independent measurements in duplicates. d Immunoblotting results of NIH-3T3 cells (EGFR 19del or EGFR 19del+G724S ) monitoring phospho-EGFR and total EGFR. Cells were treated for indicated times (0, 1, 3, 6, and 24 h) with osimertinib (1 µM) or afatinib (1 µM). HSP90 was used as loading control ( n = 3)
    Figure Legend Snippet: Kinetic evaluation of second- and third-generation EGFR TKIs against EGFR mutant proteins. a Schematic overview of two-step binding mechanism of covalent inhibitors to kinases with K i (quotient of k off and k on ) describing the reversible binding affinity and k inact describing the rate of inactivation. b Time-dependent IC 50 -determination of afatinib and osimertinib on EGFR mutant proteins. Representative curves of single measurements in duplicates are shown. c Heatmap of biochemical IC 50 -, K i -, and k inact determination of second- and third-generation EGFR TKIs against EGFR mutant proteins. Values are the mean of three independent measurements in duplicates. d Immunoblotting results of NIH-3T3 cells (EGFR 19del or EGFR 19del+G724S ) monitoring phospho-EGFR and total EGFR. Cells were treated for indicated times (0, 1, 3, 6, and 24 h) with osimertinib (1 µM) or afatinib (1 µM). HSP90 was used as loading control ( n = 3)

    Techniques Used: Mutagenesis, Binding Assay

    EGFR G724S mediates resistance to third-generation EGFR inhibitors in vitro and in vivo. a Immunoblotting results of NIH-3T3 cells (empty vector, EGFR 19del , EGFR G724S , or EGFR 19del+G724S ) monitoring phospho-EGFR and total EGFR under erlotinib treatment (24 h). HSP90 was used as loading control. b Immunoblotting of NIH-3T3 cells under osimertinib treatment (24 h) is shown. Dose–response measurement of Ba/F3 cells expressing EGFR 19del , EGFR G724S , or EGFR 19del+G724S treated for 72 h with c erlotinib or d osimertinib. Experiments were performed at least three times. e , f Graphs show tumor volume of mice injected with NIH-3T3 EGFR 19del and EGFR 19del+G724S cells treated with osimertinib (5 mg/kg, i.p., once daily). e Tumor volumes ( EGFR 19del vehicle, n = 7 mice; EGFR 19del osimertinib, n = 8 mice; EGFR 19del+G724S vehicle, n = 7 mice; EGFR 19del+G724S osimertinib, n =10 mice) were assessed for 20 days by longitudinal caliper measurements every second day following treatment initiation. f Tumor volumes were quantified after 8 days of treatment. Volume changes in the osimertinib treatment cohort (dark gray and green) were compared with the vehicle-treated control group (light gray and green). Each dot represents a single tumor per mouse. Significance is calculated by two-tailed Student’s t test, n.s.: non-significant. g Representative images of Cleaved Caspase-3 stainings. Tumors of mice bearing NIH-3T3 EGFR 19del or EGFR 19del+G724S cells were treated with vehicle solution HPMC (0, 5%) or osimertinib. Scale bar 100 μm. h Quantification of Cleaved Caspase-3 staining. Number of apoptotic cells in the osimertinib-treated cohort (dark gray and green) was compared with the vehicle-treated control group (light gray and green)
    Figure Legend Snippet: EGFR G724S mediates resistance to third-generation EGFR inhibitors in vitro and in vivo. a Immunoblotting results of NIH-3T3 cells (empty vector, EGFR 19del , EGFR G724S , or EGFR 19del+G724S ) monitoring phospho-EGFR and total EGFR under erlotinib treatment (24 h). HSP90 was used as loading control. b Immunoblotting of NIH-3T3 cells under osimertinib treatment (24 h) is shown. Dose–response measurement of Ba/F3 cells expressing EGFR 19del , EGFR G724S , or EGFR 19del+G724S treated for 72 h with c erlotinib or d osimertinib. Experiments were performed at least three times. e , f Graphs show tumor volume of mice injected with NIH-3T3 EGFR 19del and EGFR 19del+G724S cells treated with osimertinib (5 mg/kg, i.p., once daily). e Tumor volumes ( EGFR 19del vehicle, n = 7 mice; EGFR 19del osimertinib, n = 8 mice; EGFR 19del+G724S vehicle, n = 7 mice; EGFR 19del+G724S osimertinib, n =10 mice) were assessed for 20 days by longitudinal caliper measurements every second day following treatment initiation. f Tumor volumes were quantified after 8 days of treatment. Volume changes in the osimertinib treatment cohort (dark gray and green) were compared with the vehicle-treated control group (light gray and green). Each dot represents a single tumor per mouse. Significance is calculated by two-tailed Student’s t test, n.s.: non-significant. g Representative images of Cleaved Caspase-3 stainings. Tumors of mice bearing NIH-3T3 EGFR 19del or EGFR 19del+G724S cells were treated with vehicle solution HPMC (0, 5%) or osimertinib. Scale bar 100 μm. h Quantification of Cleaved Caspase-3 staining. Number of apoptotic cells in the osimertinib-treated cohort (dark gray and green) was compared with the vehicle-treated control group (light gray and green)

    Techniques Used: In Vitro, In Vivo, Plasmid Preparation, Expressing, Mouse Assay, Injection, Two Tailed Test, Staining

    Biochemical profiling of EGFR G724S . a Homogeneous time-resolved fluorescence (HTRF) is used for IC 50 -determination for EGFR 19del and EGFR 19del +G724S with representative inhibitors. Representative dose–response curves of a single measurement in duplicates are shown. b Comparison of biochemical IC 50 -values with HTRF for the three generations of EGFR TKIs against EGFR 19del+G724S . Values are the mean of three independent measurements in duplicates. c Immunoblotting results of NIH-3T3 cells (empty vector, EGFR 19del , EGFR G724S or EGFR 19del+G724S ) showing phospho-EGFR and total EGFR under afatinib treatment (24 h). HSP90 was used as loading control ( n = 3). d Dose–response measurement of Ba/F3 cells expressing EGFR 19del , EGFR G724S , or EGFR 19del+G724S treated for 72 h with afatinib. Experiments were accomplished for at least three times. e Structure of exon 20 mutant EGFR , bound to 4-anilinoquinazoline based TKI PD168393, is shown
    Figure Legend Snippet: Biochemical profiling of EGFR G724S . a Homogeneous time-resolved fluorescence (HTRF) is used for IC 50 -determination for EGFR 19del and EGFR 19del +G724S with representative inhibitors. Representative dose–response curves of a single measurement in duplicates are shown. b Comparison of biochemical IC 50 -values with HTRF for the three generations of EGFR TKIs against EGFR 19del+G724S . Values are the mean of three independent measurements in duplicates. c Immunoblotting results of NIH-3T3 cells (empty vector, EGFR 19del , EGFR G724S or EGFR 19del+G724S ) showing phospho-EGFR and total EGFR under afatinib treatment (24 h). HSP90 was used as loading control ( n = 3). d Dose–response measurement of Ba/F3 cells expressing EGFR 19del , EGFR G724S , or EGFR 19del+G724S treated for 72 h with afatinib. Experiments were accomplished for at least three times. e Structure of exon 20 mutant EGFR , bound to 4-anilinoquinazoline based TKI PD168393, is shown

    Techniques Used: Fluorescence, Plasmid Preparation, Expressing, Mutagenesis

    16) Product Images from "Integrin α6 and EGFR Signaling Converge at Mechanosensitive Calpain 2"

    Article Title: Integrin α6 and EGFR Signaling Converge at Mechanosensitive Calpain 2

    Journal: Biomaterials

    doi: 10.1016/j.biomaterials.2018.05.056

    Mechanosensitive signaling is dependent on laminin and substrate stiffness. (a) Breast cancer cells on soft gels, or any stiffness gel supplemented with EGF, were small and motile. Cells on soft gels produced their own laminin to engage α 6 , or we added it exogenously. Our data suggests this increases activation of ERK and Calpain 2, which feeds back to increase turnover of focal adhesions. (b) During adhesion to substrates, cells were more responsive to laminin on soft substrates but did not adhere as well on stiff substrates with a calpain 2 inhibitor. Cell adhesion was facilitated in the presence of EGF on both soft and stiff gels. After 24 hours on gels, calpain 2 activity decreased with increasing stiffness, while EGFR phosphorylation increased with stiffness without added EGF. Cell motility had a biphasic dependence on substrate stiffness.
    Figure Legend Snippet: Mechanosensitive signaling is dependent on laminin and substrate stiffness. (a) Breast cancer cells on soft gels, or any stiffness gel supplemented with EGF, were small and motile. Cells on soft gels produced their own laminin to engage α 6 , or we added it exogenously. Our data suggests this increases activation of ERK and Calpain 2, which feeds back to increase turnover of focal adhesions. (b) During adhesion to substrates, cells were more responsive to laminin on soft substrates but did not adhere as well on stiff substrates with a calpain 2 inhibitor. Cell adhesion was facilitated in the presence of EGF on both soft and stiff gels. After 24 hours on gels, calpain 2 activity decreased with increasing stiffness, while EGFR phosphorylation increased with stiffness without added EGF. Cell motility had a biphasic dependence on substrate stiffness.

    Techniques Used: Produced, Activation Assay, Activity Assay

    17) Product Images from "Celecoxib increases EGF signaling in colon tumor associated fibroblasts, modulating EGFR expression and degradation"

    Article Title: Celecoxib increases EGF signaling in colon tumor associated fibroblasts, modulating EGFR expression and degradation

    Journal: Oncotarget

    doi:

    Celecoxib increases EGFR mRNA and protein expression a ) Real time PCR for EGFR. Colon TAFs were treated with Celecoxib (Cel, 10μM) for 48 hours; EGF (50ng/ml) was added as indicated during the last 16h of incubation. EGFR mRNA levels were normalized against the RP2 housekeeping gene. The mean values of three independent tests are shown. b ) A western blot for EGFR under the same condition reported for Real Time PCR. c ) The relative mean intensity of bands from six independent western blots, on three different TAFs primary cell cultures, was calculated by densitometry and plotted. d ) Flow cytometric analysis of surface and total EGFR. TAFs were treated as described above. The peaks, representing EGFR expression under Celecoxib (Cel), EGF, or Celecoxib plus EGF (Cel+EGF) treatments (white peaks), were compared to EGFR levels detected in untreated controls (grey peaks). The MFI ratio (white/grey) was calculated and reported on each panel. e ) Surface and total EGFR increase induced by Celecoxib, calculated as Cel+EGF / EGF ratio of three independent flow cytometric analyses as reported in panel d. f ) Binding and internalization of biotin-EGF in colon TAFs primed or not with Celecoxib and treated with biotin-EGF (50ng/ml) for 45 or 30min respectively. The test was run in six replicates and repeated three times.
    Figure Legend Snippet: Celecoxib increases EGFR mRNA and protein expression a ) Real time PCR for EGFR. Colon TAFs were treated with Celecoxib (Cel, 10μM) for 48 hours; EGF (50ng/ml) was added as indicated during the last 16h of incubation. EGFR mRNA levels were normalized against the RP2 housekeeping gene. The mean values of three independent tests are shown. b ) A western blot for EGFR under the same condition reported for Real Time PCR. c ) The relative mean intensity of bands from six independent western blots, on three different TAFs primary cell cultures, was calculated by densitometry and plotted. d ) Flow cytometric analysis of surface and total EGFR. TAFs were treated as described above. The peaks, representing EGFR expression under Celecoxib (Cel), EGF, or Celecoxib plus EGF (Cel+EGF) treatments (white peaks), were compared to EGFR levels detected in untreated controls (grey peaks). The MFI ratio (white/grey) was calculated and reported on each panel. e ) Surface and total EGFR increase induced by Celecoxib, calculated as Cel+EGF / EGF ratio of three independent flow cytometric analyses as reported in panel d. f ) Binding and internalization of biotin-EGF in colon TAFs primed or not with Celecoxib and treated with biotin-EGF (50ng/ml) for 45 or 30min respectively. The test was run in six replicates and repeated three times.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Incubation, Western Blot, Flow Cytometry, Binding Assay

    Celecoxib retards Cathepsin-D maturation in late endosomes, causing EGFR and Rab7 accumulation a ) Cytoplasmic vesicles from TAFs treated as shown were fractioned by centrifugation (see methods) obtaining samples enriched in lysosomes (Lys: EEA1 low; Rab7, LAMP1 high; Cathepsin-D very high), late endosomes (LE: EEA1 low; Cathepsin middle; Rab7, LAMP1 high) and early endosomes (EE: EEA1 high; Rab7, LAMP1, Cathepsin-D low). Post nuclear supernatants (Pns) from untreated controls were used as loading controls. b - e ) Relative quantification of EGFR, Rab7, pro Cathepsin-D and active Cathepsin-D bands in Lys, LE and EE-enriched fractions from replicates of the test shown in panel a. f) EGFR, Rab7 and pro/active Cathepsin-D gain in the Lys, LE and EE-enriched fractions of Celecoxib-treated samples, calculated from data shown in graphs b-e as ratios against the relative controls (both untreated or EGF-treated).
    Figure Legend Snippet: Celecoxib retards Cathepsin-D maturation in late endosomes, causing EGFR and Rab7 accumulation a ) Cytoplasmic vesicles from TAFs treated as shown were fractioned by centrifugation (see methods) obtaining samples enriched in lysosomes (Lys: EEA1 low; Rab7, LAMP1 high; Cathepsin-D very high), late endosomes (LE: EEA1 low; Cathepsin middle; Rab7, LAMP1 high) and early endosomes (EE: EEA1 high; Rab7, LAMP1, Cathepsin-D low). Post nuclear supernatants (Pns) from untreated controls were used as loading controls. b - e ) Relative quantification of EGFR, Rab7, pro Cathepsin-D and active Cathepsin-D bands in Lys, LE and EE-enriched fractions from replicates of the test shown in panel a. f) EGFR, Rab7 and pro/active Cathepsin-D gain in the Lys, LE and EE-enriched fractions of Celecoxib-treated samples, calculated from data shown in graphs b-e as ratios against the relative controls (both untreated or EGF-treated).

    Techniques Used: Centrifugation

    Celecoxib slows down EGFR degradation a ) Western blot analysis of the kinetic of EGFR degradation. Colon TAFs pretreated with Celecoxib were challenged with EGF for the indicated times. The arrow indicates the band used for EGFR degradation quantification. The test was repeated twice. b ) The early endosome marker 1 (EEA1) levels were not influenced by Celecoxib pretreatment. c ) A representative image (90min EGF) of double immunofluorescence analyses: EGFR (red), EEA1 (green). Celecoxib-pretreated colon TAFs were challenged with EGF for 30, 90, 180min or 16h. Fluorescent images were acquired, with fixed expositions (EEA1-488 f1/8; EGFR-594 f1/3; DAPI f1/100), by a Leica DM-LB2 microscope equipped with I3 and M2 filters and a HCX PL Fluotar 40x non immersion optic. A 20μm scale is shown. d ) Analysis of Mander's overlay coefficients for EGFR and EEA1 on the double immunofluorescence. Six random 40x fields per condition -containing at least 12 TAFs- were analyzed (see methods). The test was repeated twice. e ) Flow cytometric analysis for EGFR expression in TAFs pretreated or not with Celecoxib and then challenged for 90 or 180min with EGF. The peaks, representing EGFR expression under EGF, or Celecoxib plus EGF (Cel+EGF) treatments (white peaks), were compared to EGFR levels detected in untreated controls (grey peaks). The MFI ratio (white/grey) was calculated and reported on each panel. The test was repeated twice.
    Figure Legend Snippet: Celecoxib slows down EGFR degradation a ) Western blot analysis of the kinetic of EGFR degradation. Colon TAFs pretreated with Celecoxib were challenged with EGF for the indicated times. The arrow indicates the band used for EGFR degradation quantification. The test was repeated twice. b ) The early endosome marker 1 (EEA1) levels were not influenced by Celecoxib pretreatment. c ) A representative image (90min EGF) of double immunofluorescence analyses: EGFR (red), EEA1 (green). Celecoxib-pretreated colon TAFs were challenged with EGF for 30, 90, 180min or 16h. Fluorescent images were acquired, with fixed expositions (EEA1-488 f1/8; EGFR-594 f1/3; DAPI f1/100), by a Leica DM-LB2 microscope equipped with I3 and M2 filters and a HCX PL Fluotar 40x non immersion optic. A 20μm scale is shown. d ) Analysis of Mander's overlay coefficients for EGFR and EEA1 on the double immunofluorescence. Six random 40x fields per condition -containing at least 12 TAFs- were analyzed (see methods). The test was repeated twice. e ) Flow cytometric analysis for EGFR expression in TAFs pretreated or not with Celecoxib and then challenged for 90 or 180min with EGF. The peaks, representing EGFR expression under EGF, or Celecoxib plus EGF (Cel+EGF) treatments (white peaks), were compared to EGFR levels detected in untreated controls (grey peaks). The MFI ratio (white/grey) was calculated and reported on each panel. The test was repeated twice.

    Techniques Used: Western Blot, Marker, Immunofluorescence, Microscopy, Flow Cytometry, Expressing

    Celecoxib affects protein turnover mimicking the inhibitors of endo-lysosome acidification a ) Western blot for EGFR and p62/SQSTM1 of colon TAFs pretreated with Celecoxib and triggered for 3 or 16h with EGF. b ) Western blot analysis of the effects of proteasome-lysosome inhibitors. 48h treatment with MG132 (proteasome inhibitor, 2μM), Bafilomycin-A1 (Baf, V-ATPase inhibitor, 25nM), NH 4 Cl (lysosomal pH -buffering molecule, 10mM) were compared to Celecoxib (10μM) as modulators of EGFR, p62, HSP70 and IkBα. c ) Relative quantification of EGFR and p62 levels from replicate tests as shown in panel b; p values were calculated as compared to untreated controls. d ) Western blot comparison of the effects of Celecoxib and lysosome acidification inhibitors. The effects of Celecoxib (10μM), Bafilomycin-A1 (2.5nM) and NH 4 Cl (2.5mM) pretreatment were tested in the absence/presence of EGF (3h) on several target proteins: EGFR, p62, IkBα, EEA1, LAMP1, Rab7, pro Cathepsin-D and Cathepsin-D. Loading controls: beta-actin (1) normalizes EEA1, p62 and Cathepsin-D; beta-actin (2) normalizes EGFR, IkBα, LAMP1 and Rab7. e ) Relative quantification of EGFR, p62, Rab7, pro and active Cathepsin-D levels from replicate tests as shown in panel d; p values defined in Materials and Methods were calculated as compared to untreated controls.
    Figure Legend Snippet: Celecoxib affects protein turnover mimicking the inhibitors of endo-lysosome acidification a ) Western blot for EGFR and p62/SQSTM1 of colon TAFs pretreated with Celecoxib and triggered for 3 or 16h with EGF. b ) Western blot analysis of the effects of proteasome-lysosome inhibitors. 48h treatment with MG132 (proteasome inhibitor, 2μM), Bafilomycin-A1 (Baf, V-ATPase inhibitor, 25nM), NH 4 Cl (lysosomal pH -buffering molecule, 10mM) were compared to Celecoxib (10μM) as modulators of EGFR, p62, HSP70 and IkBα. c ) Relative quantification of EGFR and p62 levels from replicate tests as shown in panel b; p values were calculated as compared to untreated controls. d ) Western blot comparison of the effects of Celecoxib and lysosome acidification inhibitors. The effects of Celecoxib (10μM), Bafilomycin-A1 (2.5nM) and NH 4 Cl (2.5mM) pretreatment were tested in the absence/presence of EGF (3h) on several target proteins: EGFR, p62, IkBα, EEA1, LAMP1, Rab7, pro Cathepsin-D and Cathepsin-D. Loading controls: beta-actin (1) normalizes EEA1, p62 and Cathepsin-D; beta-actin (2) normalizes EGFR, IkBα, LAMP1 and Rab7. e ) Relative quantification of EGFR, p62, Rab7, pro and active Cathepsin-D levels from replicate tests as shown in panel d; p values defined in Materials and Methods were calculated as compared to untreated controls.

    Techniques Used: Western Blot

    Related Articles

    Western Blot:

    Article Title: A gain-of-function mutant p53-HSF1 feed forward circuit governs adaptation of cancer cells to proteotoxic stress
    Article Snippet: .. Immunoblots and immunoprecipitations For immunoblots, equal total protein of cell lysates (2.5–20 μ g) were detected with antibodies to mouse p53 (FL393), human p53 (PAb1801; Santa Cruz Biotechnology), HSF1, p-Ser326 HSF1, AKT, pAKT, Erk, pErk, Hsp70, Hsp90, Hsp90α , EGFR, EGRF-Tyr845P (all Cell Signaling, Danvers, MA, USA), ErbB2, actin, GAPDH, GTS, GFP, HSc70 and HDAC1 (all Neomarkers, Fremont, CA, USA)., , SDS-PAAG gels (6%) were used to detect slower migrating HS-activated HSF1 used in experiments presented in and . ..

    Immunohistochemistry:

    Article Title: Elevated Expression of Fn14 in Non-Small Cell Lung Cancer Correlates with Activated EGFR and Promotes Tumor Cell Migration and Invasion
    Article Snippet: .. IHC analysis for Fn14 was performed using the Fn14 monoclonal antibody P4A8 (Biogen Idec, Inc., Weston, MA), as previously described. p-EGFR analysis was performed using an antibody specific for EGFR-Y1068 (Cell Signaling Technologies, Beverly, CA). .. Human NSCLC cell lines H520, H2122, A549, H1703, H358, H3255, H1975, HCC2279, and HCC827 (ATCC, Manassas, VA) were maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA), supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a 37°C, 5% CO2 atmosphere.

    Synthesized:

    Article Title: Inhibition of histone deacetylases sensitizes EGF receptor‐TK inhibitor‐resistant non‐small‐cell lung cancer cells to erlotinib in vitro and in vivo) Inhibition of histone deacetylases sensitizes EGF receptor‐TK inhibitor‐resistant non‐small‐cell lung cancer cells to erlotinib in vitro and in vivo
    Article Snippet: Erlotinib and SAHA were purchased from Selleck Chemicals (Houston, TX, USA). .. YF454A [N1‐((5‐(5‐pyrimidinyl)‐2‐thiopheneyl) methyl)‐N7‐hydroxyN1‐(4‐methoxyphenyl) heptane‐diamide] and other lead compounds were synthesized in house (Yang et al., (Ser473 (Thr202/ Tyr204 ), ERK1/2, p‐EGFR (pTyr1068 ), EGFR, p‐Her2 (Tyr1221/1222 ), Her2, p‐c‐Met(Tyr1234/1235 ), c‐Met, p‐IGF1R (pTyr1316 , cyclin D1 and E2F1 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). .. Antibodies against Cdc25A, p‐Rb (Ser807/811 ) and Rb were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

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    Cell Signaling Technology Inc anti total egfr
    Phospho-proteomics reveals that glucose withdrawal induces a distinct signature of phospho-tyrosine signaling that is associated with focal adhesions. ( A , B ) Following glucose and pyruvate starvation of U87 and U87-EGFRvIII for the indicated times, western blotting revealed activation of some but not all signaling pathways following glucose withdrawal. (A) Phospho-specific antibodies against tyrosine residues demonstrated significantly increased phosphorylation of RTKs, including <t>EGFR,</t> Met, and PDFGRβ. The non-RTK Src also showed increased active site phosphorylation. Total EGFR and actin served as equal loading controls. (B) Phospho-specific antibodies revealed increased glucose withdrawal-induced activity of all MAPK pathways tested (ppERK 1/2, pJNK and p-p38α) but not mTOR signaling (pS235/S236-S6) or <t>Akt</t> signaling (pS473-Akt). ( C – E ) Glucose withdrawal induces a signature of hyper-phosphorylation in U87 that is associated with focal adhesions. (C) Hierarchical clustering of tyrosine phosphorylation in U87 cells reveals that glucose withdrawal induces a distinct set of phospho-events. U87 cells were treated with four stimuli known to induce tyrosine phosphorylation, including (a) EGF stimulation (10 ng/ml, 5 min), (b) vanadate treatment (1 mM, 60 min), (c) H 2 O 2 (5 mM, 30 min) and (d) glucose and pyruvate withdrawal (3 h). Changes in phospho-tyrosine signaling were measured by quantitative, label-free mass spectrometry ( Rubbi et al, 2011 ) and data were hierarchically clustered. Each row of the heatmap depicts an individual phosphorylation event, and each column represents a sample as labeled. In the heatmap, red and green represent normalized levels of high and low phosphorylation, respectively. Samples were measured in technical duplicate (r1 and r2). Branches of the dendrogram associated with upregulation by glucose withdrawal and vanadate treatment are colored orange and blue, respectively. See Supplementary Table 1 for quantitative phospho-peptide data. (D) Glucose withdrawal induces increased phosphorylation of proteins known to localize to focal adhesions. Tyrosine residues on integrin β 1 (ITGB1 pY783), caveolin 1 (CAV1 pY14), and ephrin 2A (EPH2A pY588 and pY594) show dramatically increased phosphorylation in response to 3 h of glucose withdrawal. (E) Phospho-events associated with focal adhesions are enriched following glucose withdrawal in U87 cells. Phospho-peptides were ranked according to the measured log 2 fold change in phospho-tyrosine levels following glucose withdrawal and plotted on a waterfall plot, where red and green represent increased or decreased phosphorylation, respectively. Analysis of the phospho-peptides demonstrated an enrichment for proteins annotated with the GO term Focal Adhesion (GO:0005925) at the top of the ranked list (i.e., increased phosphorylation following glucose withdrawal) (permutation-based P -value=0.02). ( F ) Western blotting revealed increased FAK Y397 phosphorylation in response to glucose withdrawal. Source data is available for this figure in the Supplementary Information.
    Anti Total 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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    99
    Cell Signaling Technology Inc total egfr
    Elevated <t>EGFR</t> signaling correlates with SOX2 expression in lung tissues in vivo . A) Immunoblots of whole lung lysates of CCSP-rtTA/TetO- EGFR <t>L858R+T790M</t> mutant mice treated with DOX for 3 and 9 weeks. B) Immunohistochemical staining of CCSP-rtTA/TetO-egfr
    Total 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
    https://www.bioz.com/result/total egfr/product/Cell Signaling Technology Inc
    Average 99 stars, based on 1 article reviews
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    total egfr - by Bioz Stars, 2021-05
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    Phospho-proteomics reveals that glucose withdrawal induces a distinct signature of phospho-tyrosine signaling that is associated with focal adhesions. ( A , B ) Following glucose and pyruvate starvation of U87 and U87-EGFRvIII for the indicated times, western blotting revealed activation of some but not all signaling pathways following glucose withdrawal. (A) Phospho-specific antibodies against tyrosine residues demonstrated significantly increased phosphorylation of RTKs, including EGFR, Met, and PDFGRβ. The non-RTK Src also showed increased active site phosphorylation. Total EGFR and actin served as equal loading controls. (B) Phospho-specific antibodies revealed increased glucose withdrawal-induced activity of all MAPK pathways tested (ppERK 1/2, pJNK and p-p38α) but not mTOR signaling (pS235/S236-S6) or Akt signaling (pS473-Akt). ( C – E ) Glucose withdrawal induces a signature of hyper-phosphorylation in U87 that is associated with focal adhesions. (C) Hierarchical clustering of tyrosine phosphorylation in U87 cells reveals that glucose withdrawal induces a distinct set of phospho-events. U87 cells were treated with four stimuli known to induce tyrosine phosphorylation, including (a) EGF stimulation (10 ng/ml, 5 min), (b) vanadate treatment (1 mM, 60 min), (c) H 2 O 2 (5 mM, 30 min) and (d) glucose and pyruvate withdrawal (3 h). Changes in phospho-tyrosine signaling were measured by quantitative, label-free mass spectrometry ( Rubbi et al, 2011 ) and data were hierarchically clustered. Each row of the heatmap depicts an individual phosphorylation event, and each column represents a sample as labeled. In the heatmap, red and green represent normalized levels of high and low phosphorylation, respectively. Samples were measured in technical duplicate (r1 and r2). Branches of the dendrogram associated with upregulation by glucose withdrawal and vanadate treatment are colored orange and blue, respectively. See Supplementary Table 1 for quantitative phospho-peptide data. (D) Glucose withdrawal induces increased phosphorylation of proteins known to localize to focal adhesions. Tyrosine residues on integrin β 1 (ITGB1 pY783), caveolin 1 (CAV1 pY14), and ephrin 2A (EPH2A pY588 and pY594) show dramatically increased phosphorylation in response to 3 h of glucose withdrawal. (E) Phospho-events associated with focal adhesions are enriched following glucose withdrawal in U87 cells. Phospho-peptides were ranked according to the measured log 2 fold change in phospho-tyrosine levels following glucose withdrawal and plotted on a waterfall plot, where red and green represent increased or decreased phosphorylation, respectively. Analysis of the phospho-peptides demonstrated an enrichment for proteins annotated with the GO term Focal Adhesion (GO:0005925) at the top of the ranked list (i.e., increased phosphorylation following glucose withdrawal) (permutation-based P -value=0.02). ( F ) Western blotting revealed increased FAK Y397 phosphorylation in response to glucose withdrawal. Source data is available for this figure in the Supplementary Information.

    Journal: Molecular Systems Biology

    Article Title: Glucose deprivation activates a metabolic and signaling amplification loop leading to cell death

    doi: 10.1038/msb.2012.20

    Figure Lengend Snippet: Phospho-proteomics reveals that glucose withdrawal induces a distinct signature of phospho-tyrosine signaling that is associated with focal adhesions. ( A , B ) Following glucose and pyruvate starvation of U87 and U87-EGFRvIII for the indicated times, western blotting revealed activation of some but not all signaling pathways following glucose withdrawal. (A) Phospho-specific antibodies against tyrosine residues demonstrated significantly increased phosphorylation of RTKs, including EGFR, Met, and PDFGRβ. The non-RTK Src also showed increased active site phosphorylation. Total EGFR and actin served as equal loading controls. (B) Phospho-specific antibodies revealed increased glucose withdrawal-induced activity of all MAPK pathways tested (ppERK 1/2, pJNK and p-p38α) but not mTOR signaling (pS235/S236-S6) or Akt signaling (pS473-Akt). ( C – E ) Glucose withdrawal induces a signature of hyper-phosphorylation in U87 that is associated with focal adhesions. (C) Hierarchical clustering of tyrosine phosphorylation in U87 cells reveals that glucose withdrawal induces a distinct set of phospho-events. U87 cells were treated with four stimuli known to induce tyrosine phosphorylation, including (a) EGF stimulation (10 ng/ml, 5 min), (b) vanadate treatment (1 mM, 60 min), (c) H 2 O 2 (5 mM, 30 min) and (d) glucose and pyruvate withdrawal (3 h). Changes in phospho-tyrosine signaling were measured by quantitative, label-free mass spectrometry ( Rubbi et al, 2011 ) and data were hierarchically clustered. Each row of the heatmap depicts an individual phosphorylation event, and each column represents a sample as labeled. In the heatmap, red and green represent normalized levels of high and low phosphorylation, respectively. Samples were measured in technical duplicate (r1 and r2). Branches of the dendrogram associated with upregulation by glucose withdrawal and vanadate treatment are colored orange and blue, respectively. See Supplementary Table 1 for quantitative phospho-peptide data. (D) Glucose withdrawal induces increased phosphorylation of proteins known to localize to focal adhesions. Tyrosine residues on integrin β 1 (ITGB1 pY783), caveolin 1 (CAV1 pY14), and ephrin 2A (EPH2A pY588 and pY594) show dramatically increased phosphorylation in response to 3 h of glucose withdrawal. (E) Phospho-events associated with focal adhesions are enriched following glucose withdrawal in U87 cells. Phospho-peptides were ranked according to the measured log 2 fold change in phospho-tyrosine levels following glucose withdrawal and plotted on a waterfall plot, where red and green represent increased or decreased phosphorylation, respectively. Analysis of the phospho-peptides demonstrated an enrichment for proteins annotated with the GO term Focal Adhesion (GO:0005925) at the top of the ranked list (i.e., increased phosphorylation following glucose withdrawal) (permutation-based P -value=0.02). ( F ) Western blotting revealed increased FAK Y397 phosphorylation in response to glucose withdrawal. Source data is available for this figure in the Supplementary Information.

    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.

    Techniques: Western Blot, Activation Assay, Activity Assay, Mass Spectrometry, Labeling

    Glucose withdrawal activates a positive feedback loop resulting in supra-physiological phospho-tyrosine signaling and ROS-mediated cell death. In cells dependent on glucose for survival, glucose and pyruvate deprivation induces oxidative stress driven by NOX and mitochondria. This oxidative stress provokes a positive feedback loop in which NOX and mitochondria generate superoxide anion (O 2 − ), which dismutes to hydrogen peroxide (H 2 O 2 ) and inhibits PTPs by oxidation (e.g., PTP-1B and PTEN). Without the negative regulation of PTPs, TKs including EGFR and Src activate NOX at focal adhesions, further amplifying ROS generation. This glucose withdrawal-induced positive feedback loop results in supra-physiological levels of tyrosine phosphorylation and ROS-mediated cell death.

    Journal: Molecular Systems Biology

    Article Title: Glucose deprivation activates a metabolic and signaling amplification loop leading to cell death

    doi: 10.1038/msb.2012.20

    Figure Lengend Snippet: Glucose withdrawal activates a positive feedback loop resulting in supra-physiological phospho-tyrosine signaling and ROS-mediated cell death. In cells dependent on glucose for survival, glucose and pyruvate deprivation induces oxidative stress driven by NOX and mitochondria. This oxidative stress provokes a positive feedback loop in which NOX and mitochondria generate superoxide anion (O 2 − ), which dismutes to hydrogen peroxide (H 2 O 2 ) and inhibits PTPs by oxidation (e.g., PTP-1B and PTEN). Without the negative regulation of PTPs, TKs including EGFR and Src activate NOX at focal adhesions, further amplifying ROS generation. This glucose withdrawal-induced positive feedback loop results in supra-physiological levels of tyrosine phosphorylation and ROS-mediated 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.

    Techniques:

    Elevated EGFR signaling correlates with SOX2 expression in lung tissues in vivo . A) Immunoblots of whole lung lysates of CCSP-rtTA/TetO- EGFR L858R+T790M mutant mice treated with DOX for 3 and 9 weeks. B) Immunohistochemical staining of CCSP-rtTA/TetO-egfr

    Journal: Lung cancer (Amsterdam, Netherlands)

    Article Title: SOX2 expression is an early event in a murine model of EGFR mutant lung cancer and promotes proliferation of a subset of EGFR mutant lung adenocarcinoma cell lines

    doi: 10.1016/j.lungcan.2014.03.021

    Figure Lengend Snippet: Elevated EGFR signaling correlates with SOX2 expression in lung tissues in vivo . A) Immunoblots of whole lung lysates of CCSP-rtTA/TetO- EGFR L858R+T790M mutant mice treated with DOX for 3 and 9 weeks. B) Immunohistochemical staining of CCSP-rtTA/TetO-egfr

    Article Snippet: Primary antibodies against P-Akt, Oct4 (#2788), P-EGFR (Y1068) (#3777), total EGFR (#2232), EGFR L858R (#3197), P-stat3 Y705 (#9145), SOX2 (#3728) from Cell Signaling (Danvers, MA) and an alpha-tubulin antibody #T5168 from Sigma (St. Louis, MO) were detected using horseradish peroxidase–linked secondary antibodies and visualized with the enhanced chemiluminescent detection system (GE Healthcare Biosciences, Pittsburgh, PA).

    Techniques: Expressing, In Vivo, Western Blot, Mutagenesis, Mouse Assay, Immunohistochemistry, Staining

    A) Erlotinib alters the expression of SOX2 and OCT4 in EGFR mutant NSCLC cell lines. Immunoblot analysis of H1975 and HCC827 cells treated with varying doses of erlotinib for 3 and 9 days. C=vehicle control. B) PI3K/AKT pathway inhibitors decrease SOX2

    Journal: Lung cancer (Amsterdam, Netherlands)

    Article Title: SOX2 expression is an early event in a murine model of EGFR mutant lung cancer and promotes proliferation of a subset of EGFR mutant lung adenocarcinoma cell lines

    doi: 10.1016/j.lungcan.2014.03.021

    Figure Lengend Snippet: A) Erlotinib alters the expression of SOX2 and OCT4 in EGFR mutant NSCLC cell lines. Immunoblot analysis of H1975 and HCC827 cells treated with varying doses of erlotinib for 3 and 9 days. C=vehicle control. B) PI3K/AKT pathway inhibitors decrease SOX2

    Article Snippet: Primary antibodies against P-Akt, Oct4 (#2788), P-EGFR (Y1068) (#3777), total EGFR (#2232), EGFR L858R (#3197), P-stat3 Y705 (#9145), SOX2 (#3728) from Cell Signaling (Danvers, MA) and an alpha-tubulin antibody #T5168 from Sigma (St. Louis, MO) were detected using horseradish peroxidase–linked secondary antibodies and visualized with the enhanced chemiluminescent detection system (GE Healthcare Biosciences, Pittsburgh, PA).

    Techniques: Expressing, Mutagenesis

    EGF receptor is autophosphorylated in Caco-2 and activates downstream signalling pathways. Caco-2 cells were stimulated with 20 ng/mL EGF for the time periods indicated. Western blotting for (a) phosphorylated EGFR or total EGFR and (b) antibodies recognising signalling enzymes is shown. Cell lysate of EGF-treated A431 cells was used as positive control. α-tubulin is shown as a loading control. Densitometry was performed using Phoretix 1D analysis software against α-tubulin (for ERK, data for p42 and p44 are shown).

    Journal: BMC Cancer

    Article Title: Identification of the angiogenic gene signature induced by EGF and hypoxia in colorectal cancer

    doi: 10.1186/1471-2407-13-518

    Figure Lengend Snippet: EGF receptor is autophosphorylated in Caco-2 and activates downstream signalling pathways. Caco-2 cells were stimulated with 20 ng/mL EGF for the time periods indicated. Western blotting for (a) phosphorylated EGFR or total EGFR and (b) antibodies recognising signalling enzymes is shown. Cell lysate of EGF-treated A431 cells was used as positive control. α-tubulin is shown as a loading control. Densitometry was performed using Phoretix 1D analysis software against α-tubulin (for ERK, data for p42 and p44 are shown).

    Article Snippet: Rabbit anti-human phospho EGFR (Tyr 1068), phospho EGFR (Tyr 845), phospho p38 MAP Kinase (Thr 180/Tyr 182), phospho p44/42 MAP Kinase (Thr 202/Tyr 204), phospho-Akt (Ser 473), total EGFR, total p38 MAPK and total p44/42 MAPK were from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Western Blot, Positive Control, Software

    Role of EGFR, Akt, and mTORCs in UTI89 invasion of mouse bladder. A and B , strips of mouse transitional epithelial tissue were individually placed in wells (12-well plates) and treated or not with the indicated inhibitor for 1 h at 37 °C. UPEC (5 × 10 7 /ml) were added for additional 2 h, and tissue strips were washed three times with PBS. The tissue strips were lysed in PBS containing 0.1% (v/v) Triton X-100. For one part ( A ), cell lysates were analyzed for EGFR and Akt phosphorylation by Western blotting using the indicated antibodies. GAPDH was used as a protein loading control. For the second part ( B ), dilutions of lysates were plated on LB agar plates to allow colony growth. Colonies were counted, and data are presented as the -fold change of control values. Statistical analysis was performed by Student's t test compared with the nontreated ( NT ) control. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Uropathogenic Escherichia coli invades bladder epithelial cells by activating kinase networks in host cells

    doi: 10.1074/jbc.RA118.003499

    Figure Lengend Snippet: Role of EGFR, Akt, and mTORCs in UTI89 invasion of mouse bladder. A and B , strips of mouse transitional epithelial tissue were individually placed in wells (12-well plates) and treated or not with the indicated inhibitor for 1 h at 37 °C. UPEC (5 × 10 7 /ml) were added for additional 2 h, and tissue strips were washed three times with PBS. The tissue strips were lysed in PBS containing 0.1% (v/v) Triton X-100. For one part ( A ), cell lysates were analyzed for EGFR and Akt phosphorylation by Western blotting using the indicated antibodies. GAPDH was used as a protein loading control. For the second part ( B ), dilutions of lysates were plated on LB agar plates to allow colony growth. Colonies were counted, and data are presented as the -fold change of control values. Statistical analysis was performed by Student's t test compared with the nontreated ( NT ) control. *, p

    Article Snippet: Antibodies were obtained as follows: p-Akt (Ser-473), p-Akt (Thr-308), total Akt, p-mTOR (Ser-2448), total mTOR, p-p70S6K, GAPDH, p-EGFR (Tyr-1068), and total EGFR from Cell Signaling Technology and Alexa Fluor 568–conjugated phalloidin from Life Technologies.

    Techniques: Western Blot