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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 egfr
    Androgen receptor regulates expression of receptor tyrosine kinases in LNCaP and VCaP cells. Cells were deprived of androgens for 72 hours prior to stimulation with a synthetic androgen (R1881) for the indicated time. A) LNCaP cells were treated with a concentration gradient of R1881, protein lysates were harvested at 24 and 48 hours and samples analysed by WB. The intensity of <t>IGF-1R</t> and <t>EGFR</t> bands were determined with densitometry, normalized to actin and the protein amount in the vehicle treated sample was set to one. Experiment was repeated twice. B) LNCaP cells were stimulated with either 10 nM R1881 or vehicle and total mRNAs were collected after 18 hours. Values were first normalized to TBP and then to the vehicle treated sample. The data shown represents values obtained from a biological replicate and standard error of mean shown. C) VCaP cells were treated with 10 nM R1881 and protein lysates were harvested at the indicated time points. The intensity of the bands was determined with densitometry, normalized to GAPDH and the Western blot signal at the zero hours timepoint was set to one. D) VCaP cells were treated with 10 nM R1881 and mRNA samples were harvested 18 hours after the stimulation. The data shown represents values obtained from a biological replicate and standard standard error of mean is shown.
    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/egfr/product/Cell Signaling Technology Inc
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    egfr - by Bioz Stars, 2021-05
    99/100 stars

    Images

    1) Product Images from "N-Linked Glycosylation Supports Cross-Talk between Receptor Tyrosine Kinases and Androgen Receptor"

    Article Title: N-Linked Glycosylation Supports Cross-Talk between Receptor Tyrosine Kinases and Androgen Receptor

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0065016

    Androgen receptor regulates expression of receptor tyrosine kinases in LNCaP and VCaP cells. Cells were deprived of androgens for 72 hours prior to stimulation with a synthetic androgen (R1881) for the indicated time. A) LNCaP cells were treated with a concentration gradient of R1881, protein lysates were harvested at 24 and 48 hours and samples analysed by WB. The intensity of IGF-1R and EGFR bands were determined with densitometry, normalized to actin and the protein amount in the vehicle treated sample was set to one. Experiment was repeated twice. B) LNCaP cells were stimulated with either 10 nM R1881 or vehicle and total mRNAs were collected after 18 hours. Values were first normalized to TBP and then to the vehicle treated sample. The data shown represents values obtained from a biological replicate and standard error of mean shown. C) VCaP cells were treated with 10 nM R1881 and protein lysates were harvested at the indicated time points. The intensity of the bands was determined with densitometry, normalized to GAPDH and the Western blot signal at the zero hours timepoint was set to one. D) VCaP cells were treated with 10 nM R1881 and mRNA samples were harvested 18 hours after the stimulation. The data shown represents values obtained from a biological replicate and standard standard error of mean is shown.
    Figure Legend Snippet: Androgen receptor regulates expression of receptor tyrosine kinases in LNCaP and VCaP cells. Cells were deprived of androgens for 72 hours prior to stimulation with a synthetic androgen (R1881) for the indicated time. A) LNCaP cells were treated with a concentration gradient of R1881, protein lysates were harvested at 24 and 48 hours and samples analysed by WB. The intensity of IGF-1R and EGFR bands were determined with densitometry, normalized to actin and the protein amount in the vehicle treated sample was set to one. Experiment was repeated twice. B) LNCaP cells were stimulated with either 10 nM R1881 or vehicle and total mRNAs were collected after 18 hours. Values were first normalized to TBP and then to the vehicle treated sample. The data shown represents values obtained from a biological replicate and standard error of mean shown. C) VCaP cells were treated with 10 nM R1881 and protein lysates were harvested at the indicated time points. The intensity of the bands was determined with densitometry, normalized to GAPDH and the Western blot signal at the zero hours timepoint was set to one. D) VCaP cells were treated with 10 nM R1881 and mRNA samples were harvested 18 hours after the stimulation. The data shown represents values obtained from a biological replicate and standard standard error of mean is shown.

    Techniques Used: Expressing, Concentration Assay, Western Blot

    N-linked glycosylation is required for the processing and localization of Insulin like Growth Factor 1-Receptor. A) LNCaP cells were deprived of androgens for 72 hours, stimulated with 10 nM R1881 and protein lysates were harvested after 48 hours of the treatment. Phaseolus Vulgaris Leucoagglutinin lectin was used to enrich proteins modified via N-linked glycosylation from androgen stimulated cells. These enriched fractions were then analyzed by the means of Western blotting and blotted for IGF-1R, EGFR and ErbB2. Two different exposures of the same experiment are shown. The intensity of each band was determined with densitometry, normalized to the background and the input sample treated with vehicle was set to one. This experiment was repeated twice. B) LNCaP cells were deprived of androgens for 72 hours, stimulated with 10 nM R1881 for 24 hours either in the presence or absence of tunicamycin (5 µg/ml) and protein lysates were harvested. The intensity of the bands were determined with densitometry, normalized to actin and the vehicle treated sample was set to one. This experiment was repeated twice. C) LNCaP cells were deprived of androgens for 72 hours, stimulated with 10 nM R1881 for 48 hours and tunicamycin (5 µg/ml) was added for the last 18 hours where indicated. The intensity of the bands were determined with densitometry, normalized to actin and the vehicle treated sample was set to one. This experiment was repeated twice. D) LNCaP cells were treated as in C, protein lysates were harvested and IGF-1R antibody was used to immunoprecipitate (IP) the receptor. Membranes were probed with an antibody against IGF-1Rβ. The bands corresponding to IGF-1R pro-receptor, IGF-1Rβ subunit and IgG heavy chain are depicted. The density of the total pro-receptor and the longer forms were determined with densitometry and vehicle treated condition was set to one. This experiment was repeated twice. E) LNCaP cells were treated as in C, harvested for immunofluorescence by methanol fixation and stained for IGF-1R. Images were obtained with a confocal microscope with the same microscope settings for each condition. This experiment was repeated twice and representative images are shown.
    Figure Legend Snippet: N-linked glycosylation is required for the processing and localization of Insulin like Growth Factor 1-Receptor. A) LNCaP cells were deprived of androgens for 72 hours, stimulated with 10 nM R1881 and protein lysates were harvested after 48 hours of the treatment. Phaseolus Vulgaris Leucoagglutinin lectin was used to enrich proteins modified via N-linked glycosylation from androgen stimulated cells. These enriched fractions were then analyzed by the means of Western blotting and blotted for IGF-1R, EGFR and ErbB2. Two different exposures of the same experiment are shown. The intensity of each band was determined with densitometry, normalized to the background and the input sample treated with vehicle was set to one. This experiment was repeated twice. B) LNCaP cells were deprived of androgens for 72 hours, stimulated with 10 nM R1881 for 24 hours either in the presence or absence of tunicamycin (5 µg/ml) and protein lysates were harvested. The intensity of the bands were determined with densitometry, normalized to actin and the vehicle treated sample was set to one. This experiment was repeated twice. C) LNCaP cells were deprived of androgens for 72 hours, stimulated with 10 nM R1881 for 48 hours and tunicamycin (5 µg/ml) was added for the last 18 hours where indicated. The intensity of the bands were determined with densitometry, normalized to actin and the vehicle treated sample was set to one. This experiment was repeated twice. D) LNCaP cells were treated as in C, protein lysates were harvested and IGF-1R antibody was used to immunoprecipitate (IP) the receptor. Membranes were probed with an antibody against IGF-1Rβ. The bands corresponding to IGF-1R pro-receptor, IGF-1Rβ subunit and IgG heavy chain are depicted. The density of the total pro-receptor and the longer forms were determined with densitometry and vehicle treated condition was set to one. This experiment was repeated twice. E) LNCaP cells were treated as in C, harvested for immunofluorescence by methanol fixation and stained for IGF-1R. Images were obtained with a confocal microscope with the same microscope settings for each condition. This experiment was repeated twice and representative images are shown.

    Techniques Used: Modification, Western Blot, Immunofluorescence, Staining, Microscopy

    2) Product Images from "FAK deletion accelerates liver regeneration after two-thirds partial hepatectomy"

    Article Title: FAK deletion accelerates liver regeneration after two-thirds partial hepatectomy

    Journal: Scientific Reports

    doi: 10.1038/srep34316

    Fak deficiency accelerates proliferation of hepatocytes after PHx by enhancing activation of EGFR. (A) Left, expression of p-EGFR, EGFR and β-actin proteins in whole livers of Hep WT and Hep ∆Fak mice (pooled samples from 3 mice) 0, 1, 1.5, 2, 3, 5 and 7 days after PHx. Right, quantification of western blotting by Image J software. (B) Expression of p-MET, MET and β-actin proteins in whole livers of Hep WT and Hep ∆Fak mice (pooled samples from 3 mice) 0, 1, 1.5, 2, 3, 5 and 7 days after PHx. (C) Expression of p-EGFR, EGFR and β-actin proteins in whole livers of Hep WT and Hep ∆Fak mice treated with either vehicle or 50 mg/kg erlotinib by oral gavage daily for 3 days beginning one day before PHx. (D) Liver weight/body weight ratios were analyzed in Hep WT and Hep ∆Fak mice treated with either vehicle or 50 mg/kg erlotinib by oral gavage daily for 3 days beginning one day before PHx (n = 6). (E) Representative photomicrographs and quantification of immunohistochemistry for Ki67 in the livers of Hep WT and Hep ∆Fak mice for (D) (n = 6). (F) Representative photomicrographs and quantification of immunohistochemistry for BrdU in the livers of Hep WT and Hep ∆Fak mice for (D) (n = 6).
    Figure Legend Snippet: Fak deficiency accelerates proliferation of hepatocytes after PHx by enhancing activation of EGFR. (A) Left, expression of p-EGFR, EGFR and β-actin proteins in whole livers of Hep WT and Hep ∆Fak mice (pooled samples from 3 mice) 0, 1, 1.5, 2, 3, 5 and 7 days after PHx. Right, quantification of western blotting by Image J software. (B) Expression of p-MET, MET and β-actin proteins in whole livers of Hep WT and Hep ∆Fak mice (pooled samples from 3 mice) 0, 1, 1.5, 2, 3, 5 and 7 days after PHx. (C) Expression of p-EGFR, EGFR and β-actin proteins in whole livers of Hep WT and Hep ∆Fak mice treated with either vehicle or 50 mg/kg erlotinib by oral gavage daily for 3 days beginning one day before PHx. (D) Liver weight/body weight ratios were analyzed in Hep WT and Hep ∆Fak mice treated with either vehicle or 50 mg/kg erlotinib by oral gavage daily for 3 days beginning one day before PHx (n = 6). (E) Representative photomicrographs and quantification of immunohistochemistry for Ki67 in the livers of Hep WT and Hep ∆Fak mice for (D) (n = 6). (F) Representative photomicrographs and quantification of immunohistochemistry for BrdU in the livers of Hep WT and Hep ∆Fak mice for (D) (n = 6).

    Techniques Used: Activation Assay, Expressing, Mouse Assay, Western Blot, Software, Immunohistochemistry

    Fak deficiency increases HB-EGF and proliferation of hepatocytes after PHx by increasing the expression of TNFα. (A) TNFα mRNA (top) and protein (bottom) expression levels in the whole livers of Hep WT and Hep ∆Fak mice 0, 1, 1.5, 2, 3, 5 and 7 days after PHx (n = 6). (B) HB-EGF mRNA expression level in whole livers of Hep WT and Hep ∆Fak mice treated with either vehicle or a neutralized TNFα antibody by i.p. injection daily for 3 days starting one day prior to PHx. (C) expression of p-EGFR, EGFR and β-actin proteins in whole livers of Hep WT and Hep ∆Fak mice treated with either vehicle or a neutralized TNFα antibody by oral gavage daily for 3 days starting one day before PHx. (D) Liver weight/body weight ratios were analyzed in the Hep WT and Hep ∆Fak mice treated with either vehicle or a neutralized TNFα antibody by oral gavage daily for 3 days starting one day before PHx (n = 6). ( E) Representative photomicrographs and quantification (n = 6) of immunohistochemistry for Ki67 in the livers of Hep WT and Hep ∆Fak mice for (D) . (E) Representative photomicrographs and quantification of immunohistochemistry for BrdU in the livers of Hep WT and Hep ∆Fak mice (n = 5) for (D) .
    Figure Legend Snippet: Fak deficiency increases HB-EGF and proliferation of hepatocytes after PHx by increasing the expression of TNFα. (A) TNFα mRNA (top) and protein (bottom) expression levels in the whole livers of Hep WT and Hep ∆Fak mice 0, 1, 1.5, 2, 3, 5 and 7 days after PHx (n = 6). (B) HB-EGF mRNA expression level in whole livers of Hep WT and Hep ∆Fak mice treated with either vehicle or a neutralized TNFα antibody by i.p. injection daily for 3 days starting one day prior to PHx. (C) expression of p-EGFR, EGFR and β-actin proteins in whole livers of Hep WT and Hep ∆Fak mice treated with either vehicle or a neutralized TNFα antibody by oral gavage daily for 3 days starting one day before PHx. (D) Liver weight/body weight ratios were analyzed in the Hep WT and Hep ∆Fak mice treated with either vehicle or a neutralized TNFα antibody by oral gavage daily for 3 days starting one day before PHx (n = 6). ( E) Representative photomicrographs and quantification (n = 6) of immunohistochemistry for Ki67 in the livers of Hep WT and Hep ∆Fak mice for (D) . (E) Representative photomicrographs and quantification of immunohistochemistry for BrdU in the livers of Hep WT and Hep ∆Fak mice (n = 5) for (D) .

    Techniques Used: Expressing, Mouse Assay, Injection, Immunohistochemistry

    Fak deficiency increases EGFR activation and proliferation of hepatocytes after PHx by increasing expression of HB-EGF. (A) HB-EGF , TGFα , EGF and AREG mRNA expression levels in whole livers of Hep WT and Hep ∆Fak mice 0, 1, 1.5, 2, 3, 5 and 7 days after PHx (n = 6). (B) HB-EGF protein expression levels in whole livers of Hep WT and Hep ∆Fak mice (pooled samples from 3 mice) 0, 1, 1.5, 2, 3, 5 and 7 days after PHx (n = 6). (C) Expression of p-EGFR, EGFR and β-actin proteins in whole livers of Hep WT and Hep ∆Fak mice treated with either vehicle or CRM197 by oral gavage daily for 3 days starting one day before PHx. (D) Liver weight/body weight ratios were analyzed in Hep WT and Hep ∆Fak mice treated with either vehicle or CRM197 by oral gavage daily for 3 days starting one day before PHx (n = 6). ( E) Representative photomicrographs and quantification of immunohistochemistry for Ki67 in the livers of Hep WT and Hep ∆Fak mice (n = 6) for (D) . (F) Representative photomicrographs and quantification of immunohistochemistry for BrdU in the livers of Hep WT and Hep ∆Fak mice (n = 6) for (D) .
    Figure Legend Snippet: Fak deficiency increases EGFR activation and proliferation of hepatocytes after PHx by increasing expression of HB-EGF. (A) HB-EGF , TGFα , EGF and AREG mRNA expression levels in whole livers of Hep WT and Hep ∆Fak mice 0, 1, 1.5, 2, 3, 5 and 7 days after PHx (n = 6). (B) HB-EGF protein expression levels in whole livers of Hep WT and Hep ∆Fak mice (pooled samples from 3 mice) 0, 1, 1.5, 2, 3, 5 and 7 days after PHx (n = 6). (C) Expression of p-EGFR, EGFR and β-actin proteins in whole livers of Hep WT and Hep ∆Fak mice treated with either vehicle or CRM197 by oral gavage daily for 3 days starting one day before PHx. (D) Liver weight/body weight ratios were analyzed in Hep WT and Hep ∆Fak mice treated with either vehicle or CRM197 by oral gavage daily for 3 days starting one day before PHx (n = 6). ( E) Representative photomicrographs and quantification of immunohistochemistry for Ki67 in the livers of Hep WT and Hep ∆Fak mice (n = 6) for (D) . (F) Representative photomicrographs and quantification of immunohistochemistry for BrdU in the livers of Hep WT and Hep ∆Fak mice (n = 6) for (D) .

    Techniques Used: Activation Assay, Expressing, Mouse Assay, Immunohistochemistry

    3) Product Images from "Dual inhibition of EGFR and MET induces synthetic lethality in triple-negative breast cancer cells through downregulation of ribosomal protein S6"

    Article Title: Dual inhibition of EGFR and MET induces synthetic lethality in triple-negative breast cancer cells through downregulation of ribosomal protein S6

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2015.2982

    MSL subtype TNBC cells are resistant to either gefitinib or SU11274 in spite of high expression of EGFR and MET. (A) Cell lysates from exponentially growing cells were subjected to western blot analysis with indicated antibodies. β-actin was used as a loading control. (B and C) Cells were incubated with increasing concentrations of gefitinib (B) or SU11274 (C) for ≤72 h and the viable cells were determined by MTT cell viability assay. Data are presented as mean ± SEM from three independent experiments performed in triplicate.
    Figure Legend Snippet: MSL subtype TNBC cells are resistant to either gefitinib or SU11274 in spite of high expression of EGFR and MET. (A) Cell lysates from exponentially growing cells were subjected to western blot analysis with indicated antibodies. β-actin was used as a loading control. (B and C) Cells were incubated with increasing concentrations of gefitinib (B) or SU11274 (C) for ≤72 h and the viable cells were determined by MTT cell viability assay. Data are presented as mean ± SEM from three independent experiments performed in triplicate.

    Techniques Used: Expressing, Western Blot, Incubation, MTT Assay, Viability Assay

    4) Product Images from "The EGFR‐P38 MAPK axis up‐regulates PD‐L1 through miR‐675‐5p and down‐regulates HLA‐ABC via hexokinase‐2 in hepatocellular carcinoma cells, et al. The EGFR‐P38 MAPK axis up‐regulates PD‐L1 through miR‐675‐5p and down‐regulates HLA‐ABC via hexokinase‐2 in hepatocellular carcinoma cells"

    Article Title: The EGFR‐P38 MAPK axis up‐regulates PD‐L1 through miR‐675‐5p and down‐regulates HLA‐ABC via hexokinase‐2 in hepatocellular carcinoma cells, et al. The EGFR‐P38 MAPK axis up‐regulates PD‐L1 through miR‐675‐5p and down‐regulates HLA‐ABC via hexokinase‐2 in hepatocellular carcinoma cells

    Journal: Cancer Communications

    doi: 10.1002/cac2.12117

    EGFR activation is positively correlated with PD‐L1 expression while negatively correlated with HLA‐ABC expression in HCC tissues. The expression of p‐EGFR, PD‐L1, and HLA‐ABC in HCC tissues was detected using immunohistochemistry. The average scores of p‐EGFR, PD‐L1, and HLA‐ABC expression in HCCs were analyzed by using a modified quickscore assessment method. (A) The representative images of PD‐L1 and HLA‐ABC expression in HCC samples with low and high p‐EGFR expression. (B) The correlation among p‐EGFR, PD‐L1, and HLA‐ABC expression in HCCs were analyzed by using Pearson's correlation coefficient. Abbreviations: EGFR, epidermal growth factor receptor; PD‐L1, programmed death‐ligand 1; HLA‐ABC, human leukocyte antigen class‐A, B, C; HCC, hepatocellular carcinoma.
    Figure Legend Snippet: EGFR activation is positively correlated with PD‐L1 expression while negatively correlated with HLA‐ABC expression in HCC tissues. The expression of p‐EGFR, PD‐L1, and HLA‐ABC in HCC tissues was detected using immunohistochemistry. The average scores of p‐EGFR, PD‐L1, and HLA‐ABC expression in HCCs were analyzed by using a modified quickscore assessment method. (A) The representative images of PD‐L1 and HLA‐ABC expression in HCC samples with low and high p‐EGFR expression. (B) The correlation among p‐EGFR, PD‐L1, and HLA‐ABC expression in HCCs were analyzed by using Pearson's correlation coefficient. Abbreviations: EGFR, epidermal growth factor receptor; PD‐L1, programmed death‐ligand 1; HLA‐ABC, human leukocyte antigen class‐A, B, C; HCC, hepatocellular carcinoma.

    Techniques Used: Activation Assay, Expressing, Immunohistochemistry, Modification

    The EGFR‐P38 MAPK axis enhances the aerobic glycolysis which mediates HLA‐ABC down‐regulation in HCC cells . (A) SMMC‐7721 and HepG2 cells were pre‐treated with SB203580 (10 μmol/L) or DMSO for 6 h, and then stimulated with or without EGF (20 ng/mL) for an additional 24 h. Next, the cellular glucose uptake and lactate production were measured. Cells were pre‐treated with 2‐DG (10 μmol/L) or DMSO for 6 h, and then stimulated with or without EGF (20 ng/mL) for an additional 24 h. Next, (B) the cellular glucose uptake and lactate production were detected, (C) the cellular HLA‐B mRNA expression was detected by qRT‐PCR, and (D) the cell surface HLA‐ABC protein expression was detected by flow cytometry. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Abbreviations: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; HLA‐ABC, human leukocyte antigen class‐A, B, C; MAPK, mitogen‐activated protein kinase; DMSO, dimethylsulfoxide; 2‐DG, 2‐Deoxy‐D‐glucose.
    Figure Legend Snippet: The EGFR‐P38 MAPK axis enhances the aerobic glycolysis which mediates HLA‐ABC down‐regulation in HCC cells . (A) SMMC‐7721 and HepG2 cells were pre‐treated with SB203580 (10 μmol/L) or DMSO for 6 h, and then stimulated with or without EGF (20 ng/mL) for an additional 24 h. Next, the cellular glucose uptake and lactate production were measured. Cells were pre‐treated with 2‐DG (10 μmol/L) or DMSO for 6 h, and then stimulated with or without EGF (20 ng/mL) for an additional 24 h. Next, (B) the cellular glucose uptake and lactate production were detected, (C) the cellular HLA‐B mRNA expression was detected by qRT‐PCR, and (D) the cell surface HLA‐ABC protein expression was detected by flow cytometry. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Abbreviations: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; HLA‐ABC, human leukocyte antigen class‐A, B, C; MAPK, mitogen‐activated protein kinase; DMSO, dimethylsulfoxide; 2‐DG, 2‐Deoxy‐D‐glucose.

    Techniques Used: Expressing, Quantitative RT-PCR, Flow Cytometry

    PD‐L1 3’‐UTR is probably required for the EGFR‐P38 MAPK‐miR‐675‐5p axis‐enhanced stability of PD‐L1 mRNA . (A) SMMC‐7721 and HepG2 cells were transfected with firefly luciferase reporter constructs containing the PD‐L1 3’‐UTR fragment (pGL3‐ PD‐L1 3’‐UTR) or control vectors (pGL3‐Vector) for 24 h, and then co‐stimulated with or without EGF (20 ng/mL) for another 24 h. Next, the cellular luciferase activity was determined by dual‐luciferase reporter assay system. (B) Cells pre‐transfected with pGL3‐ PD‐L1 3’‐UTR were further treated with SB203580 (10 μmol/L) or DMSO for 6 h, and then co‐stimulated with or without EGF (20 ng/mL) for an additional 24 h. Next, the cellular luciferase activity was determined by dual‐luciferase reporter assay system. (C) Cells pre‐transfected with pGL3‐ PD‐L1 3’‐UTR were further treated with mimics of miR‐675‐5p or control mimics for 24 h, and then co‐stimulated with or without EGF (20 ng/mL) for an additional 24 h. Next, the cellular luciferase activity was determined by dual‐luciferase reporter assay system. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Abbreviations: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; PD‐L1, programmed death‐ligand 1; HLA‐ABC, human leukocyte antigen class‐A, B, C; MAPK, mitogen‐activated protein kinase; 3’‐UTR, 3'‐untranslated region; DMSO, dimethylsulfoxide; miRNA, micro RNA.
    Figure Legend Snippet: PD‐L1 3’‐UTR is probably required for the EGFR‐P38 MAPK‐miR‐675‐5p axis‐enhanced stability of PD‐L1 mRNA . (A) SMMC‐7721 and HepG2 cells were transfected with firefly luciferase reporter constructs containing the PD‐L1 3’‐UTR fragment (pGL3‐ PD‐L1 3’‐UTR) or control vectors (pGL3‐Vector) for 24 h, and then co‐stimulated with or without EGF (20 ng/mL) for another 24 h. Next, the cellular luciferase activity was determined by dual‐luciferase reporter assay system. (B) Cells pre‐transfected with pGL3‐ PD‐L1 3’‐UTR were further treated with SB203580 (10 μmol/L) or DMSO for 6 h, and then co‐stimulated with or without EGF (20 ng/mL) for an additional 24 h. Next, the cellular luciferase activity was determined by dual‐luciferase reporter assay system. (C) Cells pre‐transfected with pGL3‐ PD‐L1 3’‐UTR were further treated with mimics of miR‐675‐5p or control mimics for 24 h, and then co‐stimulated with or without EGF (20 ng/mL) for an additional 24 h. Next, the cellular luciferase activity was determined by dual‐luciferase reporter assay system. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Abbreviations: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; PD‐L1, programmed death‐ligand 1; HLA‐ABC, human leukocyte antigen class‐A, B, C; MAPK, mitogen‐activated protein kinase; 3’‐UTR, 3'‐untranslated region; DMSO, dimethylsulfoxide; miRNA, micro RNA.

    Techniques Used: Transfection, Luciferase, Construct, Plasmid Preparation, Activity Assay, Reporter Assay

    P38 MAPK mediates EGFR activation‐induced PD‐L1 up‐regulation and HLA‐ABC down‐regulation . (A) SMMC‐7721 cells were stimulated with EGF (20 ng/mL) for 12 h, then cells were collected and p‐P38 expression was detected by immunofluorescence. Scale bars: 40 μm. (B) SMMC‐7721 and HepG2 cells were stimulated with EGF (20 ng/mL) for indicated time periods (0‐150 min), then, the cells were collected to detect total P38 and phosphorylated P38 (p‐P38) expression by Western blotting. (C) Cells pre‐treated with EGFR inhibitor gefitinib for 6 h were further co‐stimulated with EGF (20 ng/mL) for an additional 4 h, then, the cells were collected to detect p‐P38, P38, p‐EGFR, and EGFR expression by Western blotting. Cells pre‐treated with P38 inhibitor SB203580 (SB2, 10 μmol/L) for 6 h were further stimulated with EGF (20 ng/mL) for 24 h, then PD‐L1 and HLA‐B transcription was detected by (D) qRT‐PCR, and PD‐L1 and HLA‐ABC protein levels were measured by (E) Western blotting and (F) flow cytometry. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Abbreviations: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; PD‐L1, programmed death‐ligand 1; HLA‐ABC, human leukocyte antigen class‐A, B, C; MAPK, mitogen‐activated protein kinase.
    Figure Legend Snippet: P38 MAPK mediates EGFR activation‐induced PD‐L1 up‐regulation and HLA‐ABC down‐regulation . (A) SMMC‐7721 cells were stimulated with EGF (20 ng/mL) for 12 h, then cells were collected and p‐P38 expression was detected by immunofluorescence. Scale bars: 40 μm. (B) SMMC‐7721 and HepG2 cells were stimulated with EGF (20 ng/mL) for indicated time periods (0‐150 min), then, the cells were collected to detect total P38 and phosphorylated P38 (p‐P38) expression by Western blotting. (C) Cells pre‐treated with EGFR inhibitor gefitinib for 6 h were further co‐stimulated with EGF (20 ng/mL) for an additional 4 h, then, the cells were collected to detect p‐P38, P38, p‐EGFR, and EGFR expression by Western blotting. Cells pre‐treated with P38 inhibitor SB203580 (SB2, 10 μmol/L) for 6 h were further stimulated with EGF (20 ng/mL) for 24 h, then PD‐L1 and HLA‐B transcription was detected by (D) qRT‐PCR, and PD‐L1 and HLA‐ABC protein levels were measured by (E) Western blotting and (F) flow cytometry. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Abbreviations: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; PD‐L1, programmed death‐ligand 1; HLA‐ABC, human leukocyte antigen class‐A, B, C; MAPK, mitogen‐activated protein kinase.

    Techniques Used: Activation Assay, Expressing, Immunofluorescence, Western Blot, Quantitative RT-PCR, Flow Cytometry

    miR‐675‐5p‐enhanced PD‐L1 mRNA stability is crucial for the EGFR‐P38 MAPK axis‐induced PD‐L1 accumulation . (A) SMMC‐7721 and HepG2 cells were pre‐stimulated with or without EGF for 24 h and further treated with actinomycin D (5 μg/mL) for 0‐120 min, next cellular PD‐L1 mRNA expression was detected by qRT‐PCR. (B) Cells were pre‐treated with SB203580 (10 μmol/L) or dimethylsulfoxide (DMSO) for 6 h, and then co‐stimulated with or without EGF for an additional 24 h, next cells were further treated with actinomycin D (5 μg/mL) for 0‐120 min, and cellular PD‐L1 mRNA expression was detected by qRT‐PCR. (C) Cells were stimulated with or without EGF (20 ng/mL) for 24 h, and then the expression of miR‐675‐5p was detected by qRT‐PCR. (D) Cells pre‐treated with SB203580 (10 μmol/L) or DMSO for 6 h were further co‐treated with or without EGF (20 ng/mL) for an additional 24 h, and then, the expression of miR‐675‐5p was detected by qRT‐PCR. (E) Cells were pre‐treated with mimics of miR‐675‐5p or control mimics for 24 h, and then co‐stimulated with or without EGF for an additional 24 h. Next, the cells were treated with actinomycin D (5 μg/mL) for additional indicated time periods (0‐120 min), and the cellular PD‐L1 mRNA expression was detected by qRT‐PCR. (F) Cells were pre‐transfected with mimics of miR‐675‐5p or control mimics for 24 h, and then further co‐stimulated with or without EGF (20 ng/mL) for an additional 24 h.Next, the expression of PD‐L1 was detected by qRT‐PCR and flow cytometry. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Abbreviations: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; PD‐L1, programmed death‐ligand 1; HLA‐ABC, human leukocyte antigen class‐A, B, C; MAPK, mitogen‐activated protein kinase.
    Figure Legend Snippet: miR‐675‐5p‐enhanced PD‐L1 mRNA stability is crucial for the EGFR‐P38 MAPK axis‐induced PD‐L1 accumulation . (A) SMMC‐7721 and HepG2 cells were pre‐stimulated with or without EGF for 24 h and further treated with actinomycin D (5 μg/mL) for 0‐120 min, next cellular PD‐L1 mRNA expression was detected by qRT‐PCR. (B) Cells were pre‐treated with SB203580 (10 μmol/L) or dimethylsulfoxide (DMSO) for 6 h, and then co‐stimulated with or without EGF for an additional 24 h, next cells were further treated with actinomycin D (5 μg/mL) for 0‐120 min, and cellular PD‐L1 mRNA expression was detected by qRT‐PCR. (C) Cells were stimulated with or without EGF (20 ng/mL) for 24 h, and then the expression of miR‐675‐5p was detected by qRT‐PCR. (D) Cells pre‐treated with SB203580 (10 μmol/L) or DMSO for 6 h were further co‐treated with or without EGF (20 ng/mL) for an additional 24 h, and then, the expression of miR‐675‐5p was detected by qRT‐PCR. (E) Cells were pre‐treated with mimics of miR‐675‐5p or control mimics for 24 h, and then co‐stimulated with or without EGF for an additional 24 h. Next, the cells were treated with actinomycin D (5 μg/mL) for additional indicated time periods (0‐120 min), and the cellular PD‐L1 mRNA expression was detected by qRT‐PCR. (F) Cells were pre‐transfected with mimics of miR‐675‐5p or control mimics for 24 h, and then further co‐stimulated with or without EGF (20 ng/mL) for an additional 24 h.Next, the expression of PD‐L1 was detected by qRT‐PCR and flow cytometry. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Abbreviations: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; PD‐L1, programmed death‐ligand 1; HLA‐ABC, human leukocyte antigen class‐A, B, C; MAPK, mitogen‐activated protein kinase.

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Flow Cytometry

    EGFR activation by EGF significantly induces PD‐L1 up‐regulation and HLA‐ABC down‐regulation . SMMC‐7721 and HepG2 cells were treated with or without EGF (20 ng/mL) for 24 h, then, the cells were collected to detect the expression of PD‐L1 and HLA‐ABC by (A) qRT‐PCR, (B) Western blotting, and (C) flow cytometry. (D) The cells were treated with the indicated doses of EGF (0‐40 ng/mL) for 24 h or treated with EGF (20 ng/mL) for the indicated time periods (0‐48 h), and then cellular PD‐L1 and HLA‐ABC expression was measured by flow cytometry. (E) SMMC‐7721 cells were pre‐treated with or without EGF (20 ng/mL) for 24 h, and then, the cells were collected and co‐incubated with CD8 + T cells. Next, the CD8 + T cell‐mediated lysis of tumor cells was detected 6 h later. (F) SMMC‐7721 cells were pre‐transfected with HLA‐B expression plasmids (pcDNA3.1‐ HLA‐B ) or control vector (pcDNA3.1) for 24 h, and a set of cells were collected for the detection of HLA‐ABC by flow cytometry. Another set of cells were further co‐stimulated with or without EGF (20 ng/mL) for another 24 h, and the cells were collected and co‐incubated with CD8 + T cells with or without the addition of anti‐PD‐L1 antibodies (αPD‐L1). Next, the CD8 + T cell‐mediated lysis of tumor cells was detected 6 h later. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Abbreviations: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; PD‐L1, programmed death‐ligand 1; HLA‐ABC, human leukocyte antigen class‐A, B, C.
    Figure Legend Snippet: EGFR activation by EGF significantly induces PD‐L1 up‐regulation and HLA‐ABC down‐regulation . SMMC‐7721 and HepG2 cells were treated with or without EGF (20 ng/mL) for 24 h, then, the cells were collected to detect the expression of PD‐L1 and HLA‐ABC by (A) qRT‐PCR, (B) Western blotting, and (C) flow cytometry. (D) The cells were treated with the indicated doses of EGF (0‐40 ng/mL) for 24 h or treated with EGF (20 ng/mL) for the indicated time periods (0‐48 h), and then cellular PD‐L1 and HLA‐ABC expression was measured by flow cytometry. (E) SMMC‐7721 cells were pre‐treated with or without EGF (20 ng/mL) for 24 h, and then, the cells were collected and co‐incubated with CD8 + T cells. Next, the CD8 + T cell‐mediated lysis of tumor cells was detected 6 h later. (F) SMMC‐7721 cells were pre‐transfected with HLA‐B expression plasmids (pcDNA3.1‐ HLA‐B ) or control vector (pcDNA3.1) for 24 h, and a set of cells were collected for the detection of HLA‐ABC by flow cytometry. Another set of cells were further co‐stimulated with or without EGF (20 ng/mL) for another 24 h, and the cells were collected and co‐incubated with CD8 + T cells with or without the addition of anti‐PD‐L1 antibodies (αPD‐L1). Next, the CD8 + T cell‐mediated lysis of tumor cells was detected 6 h later. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Abbreviations: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; PD‐L1, programmed death‐ligand 1; HLA‐ABC, human leukocyte antigen class‐A, B, C.

    Techniques Used: Activation Assay, Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Incubation, Lysis, Transfection, Plasmid Preparation

    HK2 mediates the EGFR‐P38 MAPK axis‐enhanced aerobic glycolysis and HLA‐ABC down‐regulation . (A) SMMC‐7721 and HepG2 cells were treated with or without EGF (20 ng/mL) for 24 h, and then the cellular HK2 expression was detected by qRT‐PCR and Western blotting. (B) Cells were pre‐treated with SB203580 (10 μmol/L) or DMSO for 6 h, and then stimulated with or without EGF (20 ng/mL) for an additional 24 h, next the cellular HK2 expression was detected by Western blotting. Cells were pre‐transfected with HK2‐specific siRNAs (siHK2) or control siRNAs (siNC) for 24 h, and then treated with or without EGF (20 ng/mL) for an additional 24 h. Next, the cellular glucose uptake and lactate production were detected (C), the cellular HLA‐B mRNA expression was detected by qRT‐PCR (D), and the cell surface HLA‐ABC protein expression was detected by flow cytometry (E). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Abbreviations: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; HLA‐ABC, human leukocyte antigen class‐A, B, C; MAPK, mitogen‐activated protein kinase; DMSO, dimethylsulfoxide; HK2, hexokinase‐2.
    Figure Legend Snippet: HK2 mediates the EGFR‐P38 MAPK axis‐enhanced aerobic glycolysis and HLA‐ABC down‐regulation . (A) SMMC‐7721 and HepG2 cells were treated with or without EGF (20 ng/mL) for 24 h, and then the cellular HK2 expression was detected by qRT‐PCR and Western blotting. (B) Cells were pre‐treated with SB203580 (10 μmol/L) or DMSO for 6 h, and then stimulated with or without EGF (20 ng/mL) for an additional 24 h, next the cellular HK2 expression was detected by Western blotting. Cells were pre‐transfected with HK2‐specific siRNAs (siHK2) or control siRNAs (siNC) for 24 h, and then treated with or without EGF (20 ng/mL) for an additional 24 h. Next, the cellular glucose uptake and lactate production were detected (C), the cellular HLA‐B mRNA expression was detected by qRT‐PCR (D), and the cell surface HLA‐ABC protein expression was detected by flow cytometry (E). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Abbreviations: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; HLA‐ABC, human leukocyte antigen class‐A, B, C; MAPK, mitogen‐activated protein kinase; DMSO, dimethylsulfoxide; HK2, hexokinase‐2.

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Flow Cytometry

    EGFR inhibitor gefitinib significantly abolishes EGFR activation‐induced PD‐L1 up‐regulation and HLA‐ABC down‐regulation in vitro and in vivo . SMMC‐7721 and HepG2 cells were pre‐treated with or without EGFR inhibitor gefitinib (10 μmol/L) for 6 h, and further co‐stimulated with or without EGF (20 ng/mL) for an additional 24 h. Then, cellular PD‐L1 and HLA‐ABC expression was measured by (A) qRT‐PCR, (B) Western blotting, and (C) flow cytometry. SMMC‐7721 cells with stable EGF expression (SMMC‐7721 pLV‐EGF ) and their control cells (SMMC‐7721 pLV‐null ) were previously established by infecting SMMC‐7721 cells using lentivirus with EGF expression vectors or empty vectors. Next, SMMC‐7721 pLV‐EGF and SMMC‐7721 pLV‐null cells were injected into the right flanks of nude mice to form xenograft tumors. When the tumor volume reached ∼100 mm 3 , tumor‐bearing mice were then treated with gefitinib (100 mg/kg) or 0.5% polysorbate vehicle once daily by oral administration (0.1 mL per 10 g body weight) for one week. Tumors were then collected, lysed, or digested for the detection of PD‐L1 and HLA‐ABC by (D) qRT‐PCR and (E) flow cytometry. SMMC‐7721 pLV‐EGF and SMMC‐7721 pLV‐null cells isolated from tumors treated with gefitinib or its vehicle were then co‐incubated with CD8 + T cells, and the specific lysis of tumor cells was detected 6 h later (F). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Abbreviations: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; PD‐L1, programmed death‐ligand 1; HLA‐ABC, human leukocyte antigen class‐A, B, C.
    Figure Legend Snippet: EGFR inhibitor gefitinib significantly abolishes EGFR activation‐induced PD‐L1 up‐regulation and HLA‐ABC down‐regulation in vitro and in vivo . SMMC‐7721 and HepG2 cells were pre‐treated with or without EGFR inhibitor gefitinib (10 μmol/L) for 6 h, and further co‐stimulated with or without EGF (20 ng/mL) for an additional 24 h. Then, cellular PD‐L1 and HLA‐ABC expression was measured by (A) qRT‐PCR, (B) Western blotting, and (C) flow cytometry. SMMC‐7721 cells with stable EGF expression (SMMC‐7721 pLV‐EGF ) and their control cells (SMMC‐7721 pLV‐null ) were previously established by infecting SMMC‐7721 cells using lentivirus with EGF expression vectors or empty vectors. Next, SMMC‐7721 pLV‐EGF and SMMC‐7721 pLV‐null cells were injected into the right flanks of nude mice to form xenograft tumors. When the tumor volume reached ∼100 mm 3 , tumor‐bearing mice were then treated with gefitinib (100 mg/kg) or 0.5% polysorbate vehicle once daily by oral administration (0.1 mL per 10 g body weight) for one week. Tumors were then collected, lysed, or digested for the detection of PD‐L1 and HLA‐ABC by (D) qRT‐PCR and (E) flow cytometry. SMMC‐7721 pLV‐EGF and SMMC‐7721 pLV‐null cells isolated from tumors treated with gefitinib or its vehicle were then co‐incubated with CD8 + T cells, and the specific lysis of tumor cells was detected 6 h later (F). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Abbreviations: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; PD‐L1, programmed death‐ligand 1; HLA‐ABC, human leukocyte antigen class‐A, B, C.

    Techniques Used: Activation Assay, In Vitro, In Vivo, Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Injection, Mouse Assay, Isolation, Incubation, Lysis

    5) Product Images from "The TGFα-EGFR-Akt signaling axis plays a role in enhancing proinflammatory chemokines in triple-negative breast cancer cells"

    Article Title: The TGFα-EGFR-Akt signaling axis plays a role in enhancing proinflammatory chemokines in triple-negative breast cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25389

    Involvement of Akt in regulating TGFα in TNBC cells ( A ) Effects of MK2206 (Akt inhibitor) on TGFα promoter activity in BT549 cells. ( B ) Enhanced effects of Akt1 overexpression on TGFα promoter activity in BT549 cells. ( C ) Effects of MK2206 on TGFα release from TNBC BT549, MB231 and HCC1806 cells. The asterisk ( * ) and hash ( # ) indicate a statistically significant increase and decrease ( p ≤ 0.05) by the Student's t -test or ANOVA and Tukey's pairwise comparisons, respectively. ( D ) Effects of MK2206 and TGFα on CXCL2 mRNA expression levels in BT549 cells. ( E ) Combined treatment of TGFα and MK2206 on CXCL2 mRNA expression levels in BT549 cells. ( F ) Schematic representation of the molecular mechanisms that drive the enrichment of proinflammatory chemokines in TNBC cells. Blue arrows represent autocrine and paracrine TGFα-EGFR-Akt signaling, and red arrows represent inflammatory burden. Purple arrows represent the combined effect of Akt activation and proinflammatory chemokine enrichment. RE: responsive elements.
    Figure Legend Snippet: Involvement of Akt in regulating TGFα in TNBC cells ( A ) Effects of MK2206 (Akt inhibitor) on TGFα promoter activity in BT549 cells. ( B ) Enhanced effects of Akt1 overexpression on TGFα promoter activity in BT549 cells. ( C ) Effects of MK2206 on TGFα release from TNBC BT549, MB231 and HCC1806 cells. The asterisk ( * ) and hash ( # ) indicate a statistically significant increase and decrease ( p ≤ 0.05) by the Student's t -test or ANOVA and Tukey's pairwise comparisons, respectively. ( D ) Effects of MK2206 and TGFα on CXCL2 mRNA expression levels in BT549 cells. ( E ) Combined treatment of TGFα and MK2206 on CXCL2 mRNA expression levels in BT549 cells. ( F ) Schematic representation of the molecular mechanisms that drive the enrichment of proinflammatory chemokines in TNBC cells. Blue arrows represent autocrine and paracrine TGFα-EGFR-Akt signaling, and red arrows represent inflammatory burden. Purple arrows represent the combined effect of Akt activation and proinflammatory chemokine enrichment. RE: responsive elements.

    Techniques Used: Activity Assay, Over Expression, Expressing, Activation Assay

    6) Product Images from "Feedback activation of STAT3 mediates trastuzumab resistance via upregulation of MUC1 and MUC4 expression"

    Article Title: Feedback activation of STAT3 mediates trastuzumab resistance via upregulation of MUC1 and MUC4 expression

    Journal: Oncotarget

    doi:

    Schematic presentation of the molecular mechanism of trastuzumab resistance mediated by STAT3-dependent feedback loop and corresponding targeting strategy (A) STAT3 is activated through multiple signaling pathways in our model, including SRC-dependent EGF:EGFR:STAT3 signaling, FN:EGFR:STAT3 signaling, and IL-6:JAK2:STAT3 signaling. Activated STAT3 promotes the expression of FN, EGF and IL-6 and therefore constitutes a positive feedback loop to persistently maintain STAT3 signaling, which leads to the upregulation of MUC1 and MUC4 mediating trastuzumab resistance via sustaining HER2 phosphorylation and/or blocking trastuzumab binding. (B) In STAT3 hyperactivation-mediated trastuzumab-resistant cancer cells, STAT3-specific small-molecule inhibitor S3I-201 efficiently inhibits STAT3 activation and consequent expression of MUC1 and MUC4, which recovers trastuzumab sensitivity leading to suppression of HER2 signaling, downstream AKT activity and finally apoptosis in response to trastuzumab treatment.
    Figure Legend Snippet: Schematic presentation of the molecular mechanism of trastuzumab resistance mediated by STAT3-dependent feedback loop and corresponding targeting strategy (A) STAT3 is activated through multiple signaling pathways in our model, including SRC-dependent EGF:EGFR:STAT3 signaling, FN:EGFR:STAT3 signaling, and IL-6:JAK2:STAT3 signaling. Activated STAT3 promotes the expression of FN, EGF and IL-6 and therefore constitutes a positive feedback loop to persistently maintain STAT3 signaling, which leads to the upregulation of MUC1 and MUC4 mediating trastuzumab resistance via sustaining HER2 phosphorylation and/or blocking trastuzumab binding. (B) In STAT3 hyperactivation-mediated trastuzumab-resistant cancer cells, STAT3-specific small-molecule inhibitor S3I-201 efficiently inhibits STAT3 activation and consequent expression of MUC1 and MUC4, which recovers trastuzumab sensitivity leading to suppression of HER2 signaling, downstream AKT activity and finally apoptosis in response to trastuzumab treatment.

    Techniques Used: Expressing, Blocking Assay, Binding Assay, Activation Assay, Activity Assay

    7) Product Images from "Intranasal delivery of VEGF enhances compensatory lung growth in mice"

    Article Title: Intranasal delivery of VEGF enhances compensatory lung growth in mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198700

    Lung tissue protein expression analyses. ELISA reveals increased VEGF levels in VEGF-treated lungs (A) on POD 4. However, quantitative polymerase chain reactions (qPCR) show no difference in mRNA transcript levels of VEGF, VEGFR1, or VEGFR2 between the two groups (B). Immunoblot demonstrates an increase in the levels of P-VEGFR2, VEGFR2, P-EGFR, EGFR, and heparin-binding EGF-like growth factor (HB-EGF) with VEGF treatment (C-D). Data are expressed as mean ± SE.
    Figure Legend Snippet: Lung tissue protein expression analyses. ELISA reveals increased VEGF levels in VEGF-treated lungs (A) on POD 4. However, quantitative polymerase chain reactions (qPCR) show no difference in mRNA transcript levels of VEGF, VEGFR1, or VEGFR2 between the two groups (B). Immunoblot demonstrates an increase in the levels of P-VEGFR2, VEGFR2, P-EGFR, EGFR, and heparin-binding EGF-like growth factor (HB-EGF) with VEGF treatment (C-D). Data are expressed as mean ± SE.

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Binding Assay

    8) Product Images from "Caveolin-1 regulates corneal wound healing by modulating Kir4.1 activity"

    Article Title: Caveolin-1 regulates corneal wound healing by modulating Kir4.1 activity

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00023.2016

    Knockout of Cav1 suppresses Kir4.1 and enhances the phosphorylation of EGFR Y845 in corneas. A : Western blot shows that Cav1 knockout decreased Kir4.1 expression. B : normalized expression of Kir4.1 in corneas from both Cav1 −/− and WT mice is shown ( P
    Figure Legend Snippet: Knockout of Cav1 suppresses Kir4.1 and enhances the phosphorylation of EGFR Y845 in corneas. A : Western blot shows that Cav1 knockout decreased Kir4.1 expression. B : normalized expression of Kir4.1 in corneas from both Cav1 −/− and WT mice is shown ( P

    Techniques Used: Knock-Out, Western Blot, Expressing, Mouse Assay

    Inhibition of Cav1 by siRNA or inhibition of Kir4.1 by Ba 2+ incubation enhances the phosphorylation of EGFR Y845 in human corneal epithelial cells (HCE). A , top : expression of phosphorylation of EGFR Y845 from both control and siRNA-Cav1 groups. A , bottom : total EGFR expression in the groups. B : normalized expression of EGFR Y845 was quantitated by densitometry ( P
    Figure Legend Snippet: Inhibition of Cav1 by siRNA or inhibition of Kir4.1 by Ba 2+ incubation enhances the phosphorylation of EGFR Y845 in human corneal epithelial cells (HCE). A , top : expression of phosphorylation of EGFR Y845 from both control and siRNA-Cav1 groups. A , bottom : total EGFR expression in the groups. B : normalized expression of EGFR Y845 was quantitated by densitometry ( P

    Techniques Used: Inhibition, Incubation, Expressing

    A scheme demonstrates the role of Kcnj10 in mediating wound healing in Cav1 knockout cornea. Inhibition of Cav1 suppresses Kcnj10 activity, which decreases K + conductance and thereby leads to membrane depolarization. As the consequence of depolarization, the phosphorylation of EGFR is stimulated causing EGF-like effects. Depolarization-induced EGF-like effects contribute to the accelerated corneal wound healing.
    Figure Legend Snippet: A scheme demonstrates the role of Kcnj10 in mediating wound healing in Cav1 knockout cornea. Inhibition of Cav1 suppresses Kcnj10 activity, which decreases K + conductance and thereby leads to membrane depolarization. As the consequence of depolarization, the phosphorylation of EGFR is stimulated causing EGF-like effects. Depolarization-induced EGF-like effects contribute to the accelerated corneal wound healing.

    Techniques Used: Knock-Out, Inhibition, Activity Assay

    9) Product Images from "Inhibition of the PI3K/AKT pathway potentiates cytotoxicity of EGFR kinase inhibitors in triple-negative breast cancer cells"

    Article Title: Inhibition of the PI3K/AKT pathway potentiates cytotoxicity of EGFR kinase inhibitors in triple-negative breast cancer cells

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.12046

    Combinations of PI3K/AKT inhibitors and EGFR inhibitors exert synergistic cytotoxicity in the susceptible cells. ( A ) SUM149PT cells were treated with various combinations of PI3K/AKT inhibitors and EGFR inhibitors for ∼72 hrs. ( B ) MDA-MB-468 cells were treated with gefitinib in combination with either PI-103 or MK-2206 for ∼72 hrs. ( C ) The non-susceptible cells (HS578T, MDA-MB-231, MDA-MB-436) were treated with gefitinib in combination with either PI-103 or MK-2206 for ∼72 hrs. (A–C) Viable cells were measured by MTT assay as described in Materials and methods. Data from two independent experiments performed in triplicate are shown as mean ± SEM. Abbreviations: Erlo, erlotinib; Gef, gefitinib; PI, PI-103; MK, MK-2206. * P
    Figure Legend Snippet: Combinations of PI3K/AKT inhibitors and EGFR inhibitors exert synergistic cytotoxicity in the susceptible cells. ( A ) SUM149PT cells were treated with various combinations of PI3K/AKT inhibitors and EGFR inhibitors for ∼72 hrs. ( B ) MDA-MB-468 cells were treated with gefitinib in combination with either PI-103 or MK-2206 for ∼72 hrs. ( C ) The non-susceptible cells (HS578T, MDA-MB-231, MDA-MB-436) were treated with gefitinib in combination with either PI-103 or MK-2206 for ∼72 hrs. (A–C) Viable cells were measured by MTT assay as described in Materials and methods. Data from two independent experiments performed in triplicate are shown as mean ± SEM. Abbreviations: Erlo, erlotinib; Gef, gefitinib; PI, PI-103; MK, MK-2206. * P

    Techniques Used: Multiple Displacement Amplification, MTT Assay

    10) Product Images from "YAP1 inhibition radiosensitizes triple negative breast cancer cells by targeting the DNA damage response and cell survival pathways"

    Article Title: YAP1 inhibition radiosensitizes triple negative breast cancer cells by targeting the DNA damage response and cell survival pathways

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21913

    YAP1 activity is required to sustain EGFR and PI3K/AKT signaling during radiation in TNBC cells ( A ) YAP1 shRNA or N.S. shRNA cells were irradiated and cell lysates, obtained two hours after radiation, and analyzed for pEGFR Y1068 , total EGFR, pAKT S473 , and total AKT ( B ) mRNA obtained from cells treated as described in (A) was analyzed for EGFR expression by qRT-PCR ( C ) MDA-MB-231 cells treated with 10 nM of verteporfin for 24 hours and irradiated. Lysates were prepared two hours later, and analyzed for YAP1, pEGFR Y1068 , total EGFR, pAKT S473 , and total AKT. ( D , E ) MDA-MB-231 and SUM159PT cells treated with YAP1 siRNA for 24 hours were subjected to radiation. Cells were collected 2 hours later and lysates were analyzed for YAP1, pAKT S473 , and total AKT. Actin was used as loading control. Values shown are the means + SE of three independent experiments. ** p ≤ 0.001.
    Figure Legend Snippet: YAP1 activity is required to sustain EGFR and PI3K/AKT signaling during radiation in TNBC cells ( A ) YAP1 shRNA or N.S. shRNA cells were irradiated and cell lysates, obtained two hours after radiation, and analyzed for pEGFR Y1068 , total EGFR, pAKT S473 , and total AKT ( B ) mRNA obtained from cells treated as described in (A) was analyzed for EGFR expression by qRT-PCR ( C ) MDA-MB-231 cells treated with 10 nM of verteporfin for 24 hours and irradiated. Lysates were prepared two hours later, and analyzed for YAP1, pEGFR Y1068 , total EGFR, pAKT S473 , and total AKT. ( D , E ) MDA-MB-231 and SUM159PT cells treated with YAP1 siRNA for 24 hours were subjected to radiation. Cells were collected 2 hours later and lysates were analyzed for YAP1, pAKT S473 , and total AKT. Actin was used as loading control. Values shown are the means + SE of three independent experiments. ** p ≤ 0.001.

    Techniques Used: Activity Assay, shRNA, Irradiation, Expressing, Quantitative RT-PCR, Multiple Displacement Amplification

    11) Product Images from "FGFR3-TACC3 fusion proteins act as naturally occurring drivers of tumor resistance by functionally substituting for EGFR/ERK signaling"

    Article Title: FGFR3-TACC3 fusion proteins act as naturally occurring drivers of tumor resistance by functionally substituting for EGFR/ERK signaling

    Journal: Oncogene

    doi: 10.1038/onc.2016.216

    FGFR3-TACC3 fusion proteins promote resistance to EGFR/ERBB3 blockade. ( a ) Parental FaDu cells were infected with an empty vector control lentivirus or with lentiviruses encoding wild-type FGFR3, the FGFR3-TACC3 fusion proteins (F3-T3) identified in the FaDu variants or a kinase-dead version of the V2 FGFR3-TACC3 fusion, and stable cell lines were generated. Cell lysates were prepared and subjected to western blot analysis with antibodies against FGFR3, phospho-FGFR, TACC3 or Actin. ( b ) Lysates were prepared from parental FaDu cells expressing wild-type FGFR3 or FGFR3-TACC3 fusion proteins (F3-T3) and were subjected to immunoprecipitation with anti-p-Tyr antibody 4G10 conjugated to agarose beads. The presence of FGFR3, TACC3 and Src in the immunoprecipitates was assessed by western blot analysis. ( c ) Parental FaDu cells expressing wild-type FGFR3 or FGFR3-TACC3 fusion protein (from V2 cells) were treated for 2 h with control antibody (15 μg/ml), REGN1400 (5 μg/ml) or REGN955 (10 μg/ml). Cell lysates were prepared and subjected to western blot analysis with antibodies against phospho-ERK, ERK, phospho-AKT or AKT. ( d ) Parental FaDu cells expressing wild-type FGFR3, FGFR3-TACC3 fusion proteins or kinase-dead FGFR3-TACC3 fusion were grown for 72 h in the presence of control antibody (15 μg/ml), REGN1400 (5 μg/ml), REGN955 (10 μg/ml) or the combination of REGN1400 plus REGN955. The bar graphs show the relative cell growth in each treatment group, as determined by MTS assay. Error bars show the s.d., n =8. Cell growth was compared by one-way ANOVA with Tukey's multiple comparison test (*** P
    Figure Legend Snippet: FGFR3-TACC3 fusion proteins promote resistance to EGFR/ERBB3 blockade. ( a ) Parental FaDu cells were infected with an empty vector control lentivirus or with lentiviruses encoding wild-type FGFR3, the FGFR3-TACC3 fusion proteins (F3-T3) identified in the FaDu variants or a kinase-dead version of the V2 FGFR3-TACC3 fusion, and stable cell lines were generated. Cell lysates were prepared and subjected to western blot analysis with antibodies against FGFR3, phospho-FGFR, TACC3 or Actin. ( b ) Lysates were prepared from parental FaDu cells expressing wild-type FGFR3 or FGFR3-TACC3 fusion proteins (F3-T3) and were subjected to immunoprecipitation with anti-p-Tyr antibody 4G10 conjugated to agarose beads. The presence of FGFR3, TACC3 and Src in the immunoprecipitates was assessed by western blot analysis. ( c ) Parental FaDu cells expressing wild-type FGFR3 or FGFR3-TACC3 fusion protein (from V2 cells) were treated for 2 h with control antibody (15 μg/ml), REGN1400 (5 μg/ml) or REGN955 (10 μg/ml). Cell lysates were prepared and subjected to western blot analysis with antibodies against phospho-ERK, ERK, phospho-AKT or AKT. ( d ) Parental FaDu cells expressing wild-type FGFR3, FGFR3-TACC3 fusion proteins or kinase-dead FGFR3-TACC3 fusion were grown for 72 h in the presence of control antibody (15 μg/ml), REGN1400 (5 μg/ml), REGN955 (10 μg/ml) or the combination of REGN1400 plus REGN955. The bar graphs show the relative cell growth in each treatment group, as determined by MTS assay. Error bars show the s.d., n =8. Cell growth was compared by one-way ANOVA with Tukey's multiple comparison test (*** P

    Techniques Used: Infection, Plasmid Preparation, Stable Transfection, Generated, Western Blot, Expressing, Immunoprecipitation, MTS Assay

    EGFR/ERBB3 blockade fails to inhibit ERK activation and cell growth in FaDu-resistant variant cell lines. ( a–c ) FaDu P1, V1 or V2 cells were grown for 72 h in the presence of control antibody (15 μg/ml), REGN1400 (5 μg/ml), REGN955 (10 μg/ml) or the combination of REGN1400 plus REGN955. The bar graphs show the relative cell growth in each treatment group, as determined by MTS assay. Error bars show the s.d., n =8. Cell growth was compared by one-way analysis of variance (ANOVA) with Tukey's multiple comparisons test (*** P
    Figure Legend Snippet: EGFR/ERBB3 blockade fails to inhibit ERK activation and cell growth in FaDu-resistant variant cell lines. ( a–c ) FaDu P1, V1 or V2 cells were grown for 72 h in the presence of control antibody (15 μg/ml), REGN1400 (5 μg/ml), REGN955 (10 μg/ml) or the combination of REGN1400 plus REGN955. The bar graphs show the relative cell growth in each treatment group, as determined by MTS assay. Error bars show the s.d., n =8. Cell growth was compared by one-way analysis of variance (ANOVA) with Tukey's multiple comparisons test (*** P

    Techniques Used: Activation Assay, Variant Assay, MTS Assay

    Generation of FaDu cell lines resistant to EGFR/ERBB3 blockade. ( a ) Severe combined immunodeficiency (SCID) mice bearing established FaDu tumors (~200 mm 3 in volume) were randomized and treated continuously with control antibody (12.5 mg/kg), REGN1400 (ERBB3-blocking antibody; 2.5 mg/kg), REGN955 (EGFR-blocking antibody; 10 mg/kg) or the combination of REGN1400 plus REGN955. The line graph depicts the average tumor volumes over the course of treatment. Error bars show the s.d. A tumor in a combination-treated mouse that began to regrow at ~110 days after implantation (middle panel) was harvested and fragments of this tumor were re-implanted into mice. A tumor fragment that grew rapidly in the face of REGN1400 plus REGN1955 combination treatment was harvested (top right panel shows the growth of individual re-implanted fragments) and the re-implantation and treatment procedure was repeated. Finally, a tumor growing rapidly under combined EGFR/ERBB3 blockade was harvested (bottom right panel) and used to generate a resistant cell line. ( b and c ) Cultured FaDu V1 or V2 cells were implanted into SCID mice to generate tumors. Mice bearing established tumors were randomized and treated twice per week with control antibody or Fc protein (12.5 mg/kg), REGN1400 (2.5 mg/kg), REGN955 (10 mg/kg) or the combination of REGN1400 plus REGN955. The line graphs depict the average tumor volumes over the course of treatment. Error bars show the s.d.
    Figure Legend Snippet: Generation of FaDu cell lines resistant to EGFR/ERBB3 blockade. ( a ) Severe combined immunodeficiency (SCID) mice bearing established FaDu tumors (~200 mm 3 in volume) were randomized and treated continuously with control antibody (12.5 mg/kg), REGN1400 (ERBB3-blocking antibody; 2.5 mg/kg), REGN955 (EGFR-blocking antibody; 10 mg/kg) or the combination of REGN1400 plus REGN955. The line graph depicts the average tumor volumes over the course of treatment. Error bars show the s.d. A tumor in a combination-treated mouse that began to regrow at ~110 days after implantation (middle panel) was harvested and fragments of this tumor were re-implanted into mice. A tumor fragment that grew rapidly in the face of REGN1400 plus REGN1955 combination treatment was harvested (top right panel shows the growth of individual re-implanted fragments) and the re-implantation and treatment procedure was repeated. Finally, a tumor growing rapidly under combined EGFR/ERBB3 blockade was harvested (bottom right panel) and used to generate a resistant cell line. ( b and c ) Cultured FaDu V1 or V2 cells were implanted into SCID mice to generate tumors. Mice bearing established tumors were randomized and treated twice per week with control antibody or Fc protein (12.5 mg/kg), REGN1400 (2.5 mg/kg), REGN955 (10 mg/kg) or the combination of REGN1400 plus REGN955. The line graphs depict the average tumor volumes over the course of treatment. Error bars show the s.d.

    Techniques Used: Mouse Assay, Blocking Assay, Cell Culture

    12) Product Images from "A Positive Feedback Loop of ER-?36/EGFR Promotes Malignant Growth of ER-negative Breast Cancer Cells"

    Article Title: A Positive Feedback Loop of ER-?36/EGFR Promotes Malignant Growth of ER-negative Breast Cancer Cells

    Journal: Oncogene

    doi: 10.1038/onc.2010.458

    ER-α36 interacts with the EGFR complex and mediates E2β-induced phosphorylation of Src and EGFR (a b). Co-immunoprecipitation and Western blot analysis of HA-ER-α36 and the EGFR complex in MDA-MB-231 (a) and MDA-MB-436 (b) cells. Cells transiently transfected with an expression of HA-tagged ER-α36 were lysised and the cell lysates were immunoprecipitated with pre-immune, anti-EGFR and anti-HA antibodies. The immunoprecipitates were blotted by anti-HA, anti-EGFR, anti-Shc, and anti-Src antibodies. (c). Western blot analysis of the effects of E2β 1nM) on the phosphorylation levels of EGFR-845 and Src-846 in MDA-MB-231 and MDA-MB-436 cells. The columns represent the means of three experiments; bars, SE. *, P
    Figure Legend Snippet: ER-α36 interacts with the EGFR complex and mediates E2β-induced phosphorylation of Src and EGFR (a b). Co-immunoprecipitation and Western blot analysis of HA-ER-α36 and the EGFR complex in MDA-MB-231 (a) and MDA-MB-436 (b) cells. Cells transiently transfected with an expression of HA-tagged ER-α36 were lysised and the cell lysates were immunoprecipitated with pre-immune, anti-EGFR and anti-HA antibodies. The immunoprecipitates were blotted by anti-HA, anti-EGFR, anti-Shc, and anti-Src antibodies. (c). Western blot analysis of the effects of E2β 1nM) on the phosphorylation levels of EGFR-845 and Src-846 in MDA-MB-231 and MDA-MB-436 cells. The columns represent the means of three experiments; bars, SE. *, P

    Techniques Used: Immunoprecipitation, Western Blot, Multiple Displacement Amplification, Transfection, Expressing

    13) Product Images from "ALK is a therapeutic target for lethal sepsis"

    Article Title: ALK is a therapeutic target for lethal sepsis

    Journal: Science translational medicine

    doi: 10.1126/scitranslmed.aan5689

    ALK/EGFR binding triggers AKT-dependent STING activation ( A ) Western blot analysis of indicated protein expression in iBMDMs and RAW264.7 and THP1 cells after stimulation with 3′3′-cGAMP (10 μg/ml), c-di-AMP (10 μg/ml), or DMXAA (10 μg/ml) for 16 hours. ( B ) Heatmap of RTKs phosphorylation changes in iBMDMs after 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) stimulation for 16 hours with or without pharmacologic (LDK378, 10 μM) or genetic inhibition of ALK. ( C ) Relative EGFR phosphorylation assayed in parallel to (B). ( D ) Immunoprecipitation (IP) analysis of the interaction between ALK and EGFR in iBMDMs after 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) stimulation for 16 hours with or without LDK378 (10 μM) or OSI-420 (10 μM). IB, immunoblotting. ( E ) Western blot analysis of indicated protein expression in iBMDMs after treatment with 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) for 16 hours with or without LDK378 (10 μM), OSI-420 (10 μM), or GDC-0068 (10 μM). ( F ) Western blot analysis of EGFR expression in EGFR stable knockdown iBMDMs. ( G ) Western blot analysis of indicated protein expression in EGFR-WT and EGFR-knockdown iBMDMs after stimulation with 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) for 16 hours. ( H and I ) iBMDMs were treated with 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) for 16 hours with or without LDK378 (10 μM), OSI-420 (10 μM), or GDC-0068 (10 μM), and IFNβ protein release (H) and IFNβ mRNA expression (I) were assayed (n = 3; data are means ± SD; * P
    Figure Legend Snippet: ALK/EGFR binding triggers AKT-dependent STING activation ( A ) Western blot analysis of indicated protein expression in iBMDMs and RAW264.7 and THP1 cells after stimulation with 3′3′-cGAMP (10 μg/ml), c-di-AMP (10 μg/ml), or DMXAA (10 μg/ml) for 16 hours. ( B ) Heatmap of RTKs phosphorylation changes in iBMDMs after 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) stimulation for 16 hours with or without pharmacologic (LDK378, 10 μM) or genetic inhibition of ALK. ( C ) Relative EGFR phosphorylation assayed in parallel to (B). ( D ) Immunoprecipitation (IP) analysis of the interaction between ALK and EGFR in iBMDMs after 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) stimulation for 16 hours with or without LDK378 (10 μM) or OSI-420 (10 μM). IB, immunoblotting. ( E ) Western blot analysis of indicated protein expression in iBMDMs after treatment with 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) for 16 hours with or without LDK378 (10 μM), OSI-420 (10 μM), or GDC-0068 (10 μM). ( F ) Western blot analysis of EGFR expression in EGFR stable knockdown iBMDMs. ( G ) Western blot analysis of indicated protein expression in EGFR-WT and EGFR-knockdown iBMDMs after stimulation with 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) for 16 hours. ( H and I ) iBMDMs were treated with 3′3′-cGAMP (10 μg/ml) or c-di-AMP (10 μg/ml) for 16 hours with or without LDK378 (10 μM), OSI-420 (10 μM), or GDC-0068 (10 μM), and IFNβ protein release (H) and IFNβ mRNA expression (I) were assayed (n = 3; data are means ± SD; * P

    Techniques Used: Binding Assay, Activation Assay, Western Blot, Expressing, Inhibition, Immunoprecipitation

    14) Product Images from "Activation of oral epithelial EphA2-EFGR signaling by Candida albicans virulence factors"

    Article Title: Activation of oral epithelial EphA2-EFGR signaling by Candida albicans virulence factors

    Journal: bioRxiv

    doi: 10.1101/491076

    EGFR signaling is required to induce a subset of epithelial cells inflammatory responses in vitro . (A) Effects of siRNA knockdown of EGFR and EphA2 on the production of the indicated cytokines and chemokines by OKF6/TERT-2 oral epithelial cell in response to 8 h of infection with C. albicans . (B) Effects of the EGFR inhibitor gefitinib on the epithelial cell response to C. albicans . (C) Stimulation of epithelial cells by the indicated strains of C. albicans . Box whisker plots show median, interquartile range, and range of 3 independent experiments, each performed in duplicate. The data were analyzed using the Kruskal-Wallis corrected for multiple comparisons. *, p
    Figure Legend Snippet: EGFR signaling is required to induce a subset of epithelial cells inflammatory responses in vitro . (A) Effects of siRNA knockdown of EGFR and EphA2 on the production of the indicated cytokines and chemokines by OKF6/TERT-2 oral epithelial cell in response to 8 h of infection with C. albicans . (B) Effects of the EGFR inhibitor gefitinib on the epithelial cell response to C. albicans . (C) Stimulation of epithelial cells by the indicated strains of C. albicans . Box whisker plots show median, interquartile range, and range of 3 independent experiments, each performed in duplicate. The data were analyzed using the Kruskal-Wallis corrected for multiple comparisons. *, p

    Techniques Used: In Vitro, Infection, Whisker Assay

    Pharmacological inhibition of EGFR reduces C. albicans-induced EphA2 activation during OPC. (A) Gating strategy to determine EGFR and EphA2 phosphorylation in the oral epithelial cells of mice with OPC. (B) Representative histograms of CD45 - EpCam + cells showing the effects of C. albicans infection and gefitinib (GEF) treatment on the phosphorylation of EGFR and EphA2 after 1 d of OPC. (C-D) Effects of gefitinib on the percentage of oral epithelial cells with phosphorylated EGFR (C) and EphA2 (D) in mice after 1 d of OPC. Data are combined results from 6 mice per group from a single experiment. Statistical significance was determined using the Mann-Whitney test. *, p
    Figure Legend Snippet: Pharmacological inhibition of EGFR reduces C. albicans-induced EphA2 activation during OPC. (A) Gating strategy to determine EGFR and EphA2 phosphorylation in the oral epithelial cells of mice with OPC. (B) Representative histograms of CD45 - EpCam + cells showing the effects of C. albicans infection and gefitinib (GEF) treatment on the phosphorylation of EGFR and EphA2 after 1 d of OPC. (C-D) Effects of gefitinib on the percentage of oral epithelial cells with phosphorylated EGFR (C) and EphA2 (D) in mice after 1 d of OPC. Data are combined results from 6 mice per group from a single experiment. Statistical significance was determined using the Mann-Whitney test. *, p

    Techniques Used: Inhibition, Activation Assay, Mouse Assay, Infection, MANN-WHITNEY

    Effects of C. albicans als3 Δ/Δ and ece1 Δ/Δ mutants EGFR and EphA2 activation. (A) The number of cell-associated (a measure of adherence) and endocytosed organisms (orgs) per high-power field (HPF) of the indicated C. albicans strains after 120 min of infection of OKF6/TERT-2 oral epithelial cells. (B) Extent of epithelial cell damage caused by the indicated strains after 8 h of infection. Data in (A and B) are the combined results of three experiments, each performed in triplicate. Statistical significance was determined by analysis of variance with Dunnett’s test for multiple comparisons. **, p
    Figure Legend Snippet: Effects of C. albicans als3 Δ/Δ and ece1 Δ/Δ mutants EGFR and EphA2 activation. (A) The number of cell-associated (a measure of adherence) and endocytosed organisms (orgs) per high-power field (HPF) of the indicated C. albicans strains after 120 min of infection of OKF6/TERT-2 oral epithelial cells. (B) Extent of epithelial cell damage caused by the indicated strains after 8 h of infection. Data in (A and B) are the combined results of three experiments, each performed in triplicate. Statistical significance was determined by analysis of variance with Dunnett’s test for multiple comparisons. **, p

    Techniques Used: Activation Assay, Infection

    EGFR activity is required for sustained EphA2 phosphorylation. (A and B) Immunoblots showing effects of ephrin A1-Fc (EFNA1-Fc) or yeast-phase C. albicans SC5314 (Ca) on the phosphorylation of ephrin type-A receptor 2 (EphA2) in OKF6/TERT-2 oral epithelial cells after stimulation for 15 min (A) and 60 min (B) post-infection (p. i.). Results are representative of 3 independent experiments. Densitometric quantification of all 3 immunoblots is shown in S1 Fig. (C and D) Effects of epidermal growth factor (EGFR) siRNA (C) and the EGFR kinase inhibitor gefitinib (GEF) (D) on the time course of EphA2 and EGFR phosphorylation in oral epithelial cells infected with C. albicans . Results are representative of 3 independent experiments. Densitometric quantification of all 3 immunoblots such as the one in Fig. 1C is shown in S1 Fig. (E) Densitometric quantification of all 3 immunoblots such as the one in Fig. 3D . (F) Lysates of oral epithelial cells infected with C. albicans for 30 and 90 min were immunoprecipitated (IP) with antibodies against EphA2 (left) and EGFR (right), after which EphA2 and EGFR were detected by immunoblotting (Top). Immunoblots of lysates prior to immunoprecipitation, demonstrating equal amounts of input protein (Bottom). (G) Densitometric analysis of 3 independent immunoblots such as the ones shown in (F). Results are mean ± SD. Statistical significance relative to uninfected control cells was analyzed by the Student’s t test assuming unequal variances. **, p
    Figure Legend Snippet: EGFR activity is required for sustained EphA2 phosphorylation. (A and B) Immunoblots showing effects of ephrin A1-Fc (EFNA1-Fc) or yeast-phase C. albicans SC5314 (Ca) on the phosphorylation of ephrin type-A receptor 2 (EphA2) in OKF6/TERT-2 oral epithelial cells after stimulation for 15 min (A) and 60 min (B) post-infection (p. i.). Results are representative of 3 independent experiments. Densitometric quantification of all 3 immunoblots is shown in S1 Fig. (C and D) Effects of epidermal growth factor (EGFR) siRNA (C) and the EGFR kinase inhibitor gefitinib (GEF) (D) on the time course of EphA2 and EGFR phosphorylation in oral epithelial cells infected with C. albicans . Results are representative of 3 independent experiments. Densitometric quantification of all 3 immunoblots such as the one in Fig. 1C is shown in S1 Fig. (E) Densitometric quantification of all 3 immunoblots such as the one in Fig. 3D . (F) Lysates of oral epithelial cells infected with C. albicans for 30 and 90 min were immunoprecipitated (IP) with antibodies against EphA2 (left) and EGFR (right), after which EphA2 and EGFR were detected by immunoblotting (Top). Immunoblots of lysates prior to immunoprecipitation, demonstrating equal amounts of input protein (Bottom). (G) Densitometric analysis of 3 independent immunoblots such as the ones shown in (F). Results are mean ± SD. Statistical significance relative to uninfected control cells was analyzed by the Student’s t test assuming unequal variances. **, p

    Techniques Used: Activity Assay, Western Blot, Infection, Immunoprecipitation

    15) Product Images from "In Utero Exposure to Bisphenol a Promotes Mammary Tumor Risk in MMTV-Erbb2 Transgenic Mice Through the Induction of ER-erbB2 Crosstalk"

    Article Title: In Utero Exposure to Bisphenol a Promotes Mammary Tumor Risk in MMTV-Erbb2 Transgenic Mice Through the Induction of ER-erbB2 Crosstalk

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21093095

    In utero exposure to low dose BPA induces ER-RTK crosstalk in mammary tissues of FVB/N mice. Pregnant FVB/N mice were treated with vehicle (FVB/Con) or 500 ng/kg BW of BPA (FVB/BP) via subcutaneous injection, daily between GD 11 and 19. The lysate was prepared from the mammary tissues of female offspring at PND 70, followed by Western blot analysis. Total and phosphorylated protein levels of indicated key regulators in ER and EGFR/erbB2 signaling pathways were examined.
    Figure Legend Snippet: In utero exposure to low dose BPA induces ER-RTK crosstalk in mammary tissues of FVB/N mice. Pregnant FVB/N mice were treated with vehicle (FVB/Con) or 500 ng/kg BW of BPA (FVB/BP) via subcutaneous injection, daily between GD 11 and 19. The lysate was prepared from the mammary tissues of female offspring at PND 70, followed by Western blot analysis. Total and phosphorylated protein levels of indicated key regulators in ER and EGFR/erbB2 signaling pathways were examined.

    Techniques Used: In Utero, Mouse Assay, Injection, Western Blot

    In utero exposure to low dose BPA induces concurrent activation of ER and EGFR/erbB2 pathways. ( A ) Western blot analysis of total and phosphorylated protein levels of indicated key regulators in ER and EGFR/erbB2 signaling pathways. Protein lysates were extracted from the mammary tissues of mice at PND 70 with different in utero BPA exposures, as detailed in the methods. ( B ) mRNA levels of genes involved in ER and RTK signaling in the mammary tissues of PND 70 mice with in utero exposure to indicated doses of BPA were quantified using real-time PCR. Each group was based on three samples from different mice with the same treatment (* p
    Figure Legend Snippet: In utero exposure to low dose BPA induces concurrent activation of ER and EGFR/erbB2 pathways. ( A ) Western blot analysis of total and phosphorylated protein levels of indicated key regulators in ER and EGFR/erbB2 signaling pathways. Protein lysates were extracted from the mammary tissues of mice at PND 70 with different in utero BPA exposures, as detailed in the methods. ( B ) mRNA levels of genes involved in ER and RTK signaling in the mammary tissues of PND 70 mice with in utero exposure to indicated doses of BPA were quantified using real-time PCR. Each group was based on three samples from different mice with the same treatment (* p

    Techniques Used: In Utero, Activation Assay, Western Blot, Mouse Assay, Real-time Polymerase Chain Reaction

    16) Product Images from "Vascular remodeling by placenta-derived mesenchymal stem cells restores ovarian function in ovariectomized rat model via the VEGF pathway"

    Article Title: Vascular remodeling by placenta-derived mesenchymal stem cells restores ovarian function in ovariectomized rat model via the VEGF pathway

    Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

    doi: 10.1038/s41374-020-00513-1

    Upregulation of folliculogenic factors at the mRNA and protein levels after PD-MSC transplantation. a mRNA expression levels of Nanos3, Lhx8, Lin28α, Nobox, Bmp15, and Egfr were decreased in the NTx groups compared to the normal group and increased in the Tx groups compared to the NTx groups with some exceptions. b Western blot analysis showed an overall increase in the expression level in the Tx groups compared to the NTx groups with some exceptions. c Immunohistochemistry for LHX8 showed higher folliculogenic activity after Tx. PD-MSCs, treatment group. *vs. the normal group ( P
    Figure Legend Snippet: Upregulation of folliculogenic factors at the mRNA and protein levels after PD-MSC transplantation. a mRNA expression levels of Nanos3, Lhx8, Lin28α, Nobox, Bmp15, and Egfr were decreased in the NTx groups compared to the normal group and increased in the Tx groups compared to the NTx groups with some exceptions. b Western blot analysis showed an overall increase in the expression level in the Tx groups compared to the NTx groups with some exceptions. c Immunohistochemistry for LHX8 showed higher folliculogenic activity after Tx. PD-MSCs, treatment group. *vs. the normal group ( P

    Techniques Used: Transplantation Assay, Expressing, Western Blot, Immunohistochemistry, Activity Assay

    17) Product Images from "MicroRNA-374a Promotes Hepatocellular Carcinoma Cell Proliferation by Targeting Mitogen-Inducible Gene 6 (MIG-6)"

    Article Title: MicroRNA-374a Promotes Hepatocellular Carcinoma Cell Proliferation by Targeting Mitogen-Inducible Gene 6 (MIG-6)

    Journal: Oncology Research

    doi: 10.3727/096504017X15000784459799

    miR-374a activates epidermal growth factor receptor (EGFR) and phosphoinositol 3-kinase/protein kinase B (AKT)/extracellular signal-regulated kinase (ERK) signaling pathways by regulation of MIG-6 in HepG2 cells. miR-374a mimic, pc-MIG-6, and their corresponding controls were transfected into HepG2 cells. (A) The protein levels of p-EGFR, EGFR, p-AKT, and p-ERK1/2 were measured by Western blot. (B) The mRNA expressions of p-EGFR, EGFR, p-AKT, and p-ERK1/2 were determined by RT-PCR. * p
    Figure Legend Snippet: miR-374a activates epidermal growth factor receptor (EGFR) and phosphoinositol 3-kinase/protein kinase B (AKT)/extracellular signal-regulated kinase (ERK) signaling pathways by regulation of MIG-6 in HepG2 cells. miR-374a mimic, pc-MIG-6, and their corresponding controls were transfected into HepG2 cells. (A) The protein levels of p-EGFR, EGFR, p-AKT, and p-ERK1/2 were measured by Western blot. (B) The mRNA expressions of p-EGFR, EGFR, p-AKT, and p-ERK1/2 were determined by RT-PCR. * p

    Techniques Used: Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction

    18) Product Images from "SH3KBP1 Promotes Glioblastoma Tumorigenesis by Activating EGFR Signaling"

    Article Title: SH3KBP1 Promotes Glioblastoma Tumorigenesis by Activating EGFR Signaling

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2020.583984

    SH3KBP1 directly and physically interacts with and activates epidermal growth factor receptor (EGFR) signaling. (A) SH3KBP1 physically interacts with EGFR determined by Co-IP assay. (B) EGFR physically interacts with SH3KBP1 determined by Co-IP assay. (C) Confocal analysis of the co-localization of SH3KBP1 and EGFR in U87 cells. Cells were stained with SH3KBP1 (red), EGFR antibody (green) and merged images are shown. Scale bar: 50 µm. (D) Quantitative data for (C) (n=6). (E) Depletion of SH3KBP1 significantly inhibits EGFR signaling downstream genes expression. (F) Quantitative data for (E) . *, ** and *** indicate as p
    Figure Legend Snippet: SH3KBP1 directly and physically interacts with and activates epidermal growth factor receptor (EGFR) signaling. (A) SH3KBP1 physically interacts with EGFR determined by Co-IP assay. (B) EGFR physically interacts with SH3KBP1 determined by Co-IP assay. (C) Confocal analysis of the co-localization of SH3KBP1 and EGFR in U87 cells. Cells were stained with SH3KBP1 (red), EGFR antibody (green) and merged images are shown. Scale bar: 50 µm. (D) Quantitative data for (C) (n=6). (E) Depletion of SH3KBP1 significantly inhibits EGFR signaling downstream genes expression. (F) Quantitative data for (E) . *, ** and *** indicate as p

    Techniques Used: Co-Immunoprecipitation Assay, Staining, Expressing

    19) Product Images from "Deoxycholic acid activates epidermal growth factor receptor and promotes intestinal carcinogenesis by ADAM17‐dependent ligand release, et al. Deoxycholic acid activates epidermal growth factor receptor and promotes intestinal carcinogenesis by ADAM17‐dependent ligand release"

    Article Title: Deoxycholic acid activates epidermal growth factor receptor and promotes intestinal carcinogenesis by ADAM17‐dependent ligand release, et al. Deoxycholic acid activates epidermal growth factor receptor and promotes intestinal carcinogenesis by ADAM17‐dependent ligand release

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.13709

    ADAM ‐17/ EGFR signalling axis was activated in intestinal tumours of deoxycholic acid–treated Apc min/+ mice. A, Immunohistochemistry showed that DCA treatment increased the percentage of positive cells of ADAM ‐17 in intestinal tumour in Apc min/+ mice. B, DCA treatment increased the percentage of positive cells of amphiregulin in intestinal tumour in Apc min/+ mice. C,D, Phosphorylation of epidermal growth factor receptor ( EGFR ) and Akt in intestinal tumours was up‐regulated after DCA treatment. E, Western blot analysis showed that DCA increased the phosphorylation of EGFR and Akt in Apc min/+ mice. DCA , deoxycholic acid. Scale bar: 20 μm. ** P
    Figure Legend Snippet: ADAM ‐17/ EGFR signalling axis was activated in intestinal tumours of deoxycholic acid–treated Apc min/+ mice. A, Immunohistochemistry showed that DCA treatment increased the percentage of positive cells of ADAM ‐17 in intestinal tumour in Apc min/+ mice. B, DCA treatment increased the percentage of positive cells of amphiregulin in intestinal tumour in Apc min/+ mice. C,D, Phosphorylation of epidermal growth factor receptor ( EGFR ) and Akt in intestinal tumours was up‐regulated after DCA treatment. E, Western blot analysis showed that DCA increased the phosphorylation of EGFR and Akt in Apc min/+ mice. DCA , deoxycholic acid. Scale bar: 20 μm. ** P

    Techniques Used: Mouse Assay, Immunohistochemistry, Western Blot

    Deoxycholic acid activated epidermal growth factor receptor ( EGFR ) in intestinal tumour cells. A,C, Protein levels of phosphorylation and total levels of EGFR and Akt expressions in Immorto‐Min colonic epithelial cell line or human colorectal cancer cell line cells were analysed by Western blot along with time variation, using β‐actin as internal control. B,D, Proteins were quantified by densitometry using an Imaging processor program (ImageJ). DCA , deoxycholic acid. *, P
    Figure Legend Snippet: Deoxycholic acid activated epidermal growth factor receptor ( EGFR ) in intestinal tumour cells. A,C, Protein levels of phosphorylation and total levels of EGFR and Akt expressions in Immorto‐Min colonic epithelial cell line or human colorectal cancer cell line cells were analysed by Western blot along with time variation, using β‐actin as internal control. B,D, Proteins were quantified by densitometry using an Imaging processor program (ImageJ). DCA , deoxycholic acid. *, P

    Techniques Used: Western Blot, Imaging

    Model of ADAM ‐17/ EGFR signalling axis activation induced by deoxycholic acid in intestinal carcinogenesis. DCA stimulates ADAM ‐17 activation and AREG release, which is required for EGF receptor activation, EGFR /Akt signalling pathway activation and apoptosis resistance in intestinal tumour cells. ADAM ‐17, a disintegrin and metalloprotease domain‐containing protein 17; AREG , amphiregulin; DCA , deoxycholic acid; EGFR , epidermal growth factor receptor [Colour figure can be viewed at http://wileyonlinelibrary.com ]
    Figure Legend Snippet: Model of ADAM ‐17/ EGFR signalling axis activation induced by deoxycholic acid in intestinal carcinogenesis. DCA stimulates ADAM ‐17 activation and AREG release, which is required for EGF receptor activation, EGFR /Akt signalling pathway activation and apoptosis resistance in intestinal tumour cells. ADAM ‐17, a disintegrin and metalloprotease domain‐containing protein 17; AREG , amphiregulin; DCA , deoxycholic acid; EGFR , epidermal growth factor receptor [Colour figure can be viewed at http://wileyonlinelibrary.com ]

    Techniques Used: Activation Assay

    20) Product Images from "Bidirectional alteration of Cav-1 expression is associated with mitogenic conversion of its function in gastric tumor progression"

    Article Title: Bidirectional alteration of Cav-1 expression is associated with mitogenic conversion of its function in gastric tumor progression

    Journal: BMC Cancer

    doi: 10.1186/s12885-017-3770-y

    Opposite effects of low and high Cav-1 on inhibitory phosphorylation of RAF. a Cav-1 inhibition of EGFR stability. WT-Cav-1- or shCav-1-expressing cells were exposed to EGF (10 ng/ml) and EGFR level was determined using an immunoblot assay. b Effect of Cav-1 on Ras activity. GTP-Ras levels were measured using Ras activity assay. c , d Opposite effects of Cav-1 on EGF-induced MEK1/2 phosphorylation. e , f Effect of Cav-1 on EGF-induced RAF phosphorylation. Stimulatory phosphorylation of A-RAF (P-S299), B-RAF (P-S445) and C-RAF (P-S339) and inhibitory phosphorylation of C-RAF (P-S289/296/301) were detected using antibodies specific to phospho-RAF isoforms
    Figure Legend Snippet: Opposite effects of low and high Cav-1 on inhibitory phosphorylation of RAF. a Cav-1 inhibition of EGFR stability. WT-Cav-1- or shCav-1-expressing cells were exposed to EGF (10 ng/ml) and EGFR level was determined using an immunoblot assay. b Effect of Cav-1 on Ras activity. GTP-Ras levels were measured using Ras activity assay. c , d Opposite effects of Cav-1 on EGF-induced MEK1/2 phosphorylation. e , f Effect of Cav-1 on EGF-induced RAF phosphorylation. Stimulatory phosphorylation of A-RAF (P-S299), B-RAF (P-S445) and C-RAF (P-S339) and inhibitory phosphorylation of C-RAF (P-S289/296/301) were detected using antibodies specific to phospho-RAF isoforms

    Techniques Used: Inhibition, Expressing, Activity Assay

    21) Product Images from "Discovery of a novel third-generation EGFR inhibitor and identification of a potential combination strategy to overcome resistance"

    Article Title: Discovery of a novel third-generation EGFR inhibitor and identification of a potential combination strategy to overcome resistance

    Journal: Molecular Cancer

    doi: 10.1186/s12943-020-01202-9

    Effect of ASK120067 on cancer cells harboring mutant or wild-type EGFR. a and b ASK120067 or osimertinib inhibited the phosphorylation of EGFR at Tyrosine residue 1068 and its downstream signaling proteins AKT and ERK in NCI-H1975 cells (EGFR L 858 R / T 790 M ) a while showing less activity against the activation of EGFR and its downstream signaling in A431 cells expressing EGFR W T b . c Apoptosis of NCI-H1975 cells was evaluated by flow cytometry after treatment with increasing concentrations of ASK120067 for 24 to 72 h. Data are plotted as the mean ± SEM, and significance of differences was determined by Student’s t test ( ∗ p
    Figure Legend Snippet: Effect of ASK120067 on cancer cells harboring mutant or wild-type EGFR. a and b ASK120067 or osimertinib inhibited the phosphorylation of EGFR at Tyrosine residue 1068 and its downstream signaling proteins AKT and ERK in NCI-H1975 cells (EGFR L 858 R / T 790 M ) a while showing less activity against the activation of EGFR and its downstream signaling in A431 cells expressing EGFR W T b . c Apoptosis of NCI-H1975 cells was evaluated by flow cytometry after treatment with increasing concentrations of ASK120067 for 24 to 72 h. Data are plotted as the mean ± SEM, and significance of differences was determined by Student’s t test ( ∗ p

    Techniques Used: Mutagenesis, Activity Assay, Activation Assay, Expressing, Flow Cytometry

    ASK120067 exerts in vivo antitumor activity against EGFR-mutant tumor xenograft models, and proof-of-concept clinical studies validate ASK120067 as an EGFR T 790 M inhibitor. a Antitumor activity of ASK120067 in the NCI-H1975 lung cancer xenograft model following 21 days of daily treatment with ASK120067 at doses of 1, 5, and 10 mg/kg/qd, with osimertinib (10 mg/kg/qd) as a positive control. b The phosphorylation of EGFR and AKT in tumor tissues of the NCI-H1975 xenograft model were evaluated by IHC staining after 21 days of treatment with ASK120067 or vehicle control and are presented by quantitative analysis. c Antitumor efficacy of ASK120067 in a PC-9 lung cancer xenograft model. d Antitumor activity of ASK120067 in the A431 epidermoid carcinoma xenograft model. e Antitumor efficacy of ASK120067 in PDX models harboring the EGFR L 858 R / T 790 M mutation. f The expression of phosphorylated EGFR and Ki-67 in PDX xenograft tumor tissues was evaluated by IHC staining and is presented by quantitative analysis. Data are presented as the mean ± SEM, and the significance of differences was determined by Student’s t test ( ∗ p
    Figure Legend Snippet: ASK120067 exerts in vivo antitumor activity against EGFR-mutant tumor xenograft models, and proof-of-concept clinical studies validate ASK120067 as an EGFR T 790 M inhibitor. a Antitumor activity of ASK120067 in the NCI-H1975 lung cancer xenograft model following 21 days of daily treatment with ASK120067 at doses of 1, 5, and 10 mg/kg/qd, with osimertinib (10 mg/kg/qd) as a positive control. b The phosphorylation of EGFR and AKT in tumor tissues of the NCI-H1975 xenograft model were evaluated by IHC staining after 21 days of treatment with ASK120067 or vehicle control and are presented by quantitative analysis. c Antitumor efficacy of ASK120067 in a PC-9 lung cancer xenograft model. d Antitumor activity of ASK120067 in the A431 epidermoid carcinoma xenograft model. e Antitumor efficacy of ASK120067 in PDX models harboring the EGFR L 858 R / T 790 M mutation. f The expression of phosphorylated EGFR and Ki-67 in PDX xenograft tumor tissues was evaluated by IHC staining and is presented by quantitative analysis. Data are presented as the mean ± SEM, and the significance of differences was determined by Student’s t test ( ∗ p

    Techniques Used: In Vivo, Activity Assay, Mutagenesis, Positive Control, Immunohistochemistry, Staining, Expressing

    22) Product Images from "Inhibition of lung tumor growth by targeting EGFR/VEGFR-Akt/NF-κB pathways with novel theanine derivatives"

    Article Title: Inhibition of lung tumor growth by targeting EGFR/VEGFR-Akt/NF-κB pathways with novel theanine derivatives

    Journal: Oncotarget

    doi:

    Effects of TFC and TNC on protein expressions and/or phosphorylation of cyclin D1 (A), VEGFR1 (B), VEGFR2 (C), pEGFR/EGFR (D), pAkt/Akt (E), and NF-κB (F) in highly metastatic LLC cells The cells were treated for 48 h with the indicated concentrations of TFC and TNC (TFC16/TNC16–TFC250/TNC250: 0.016–0.25 mM), Ly (0.016 mM) and Bay (0.0032 mM). The protein expressions were analyzed by Western Blotting. The optical density (OD) of the band is normalized with respective β-actin and is expressed as relative optical density (OD). The OD value of the band shown as mean ± SD is relative to that of the control (DMSO vehicle) designated as 100%. Bay and Ly are the inhibitors of NF- κ B and PI3K/Akt, respectively. For one experiment, 3 assays were carried out and only one set of gels is shown. Values with different letters (a–g) differ significantly ( P
    Figure Legend Snippet: Effects of TFC and TNC on protein expressions and/or phosphorylation of cyclin D1 (A), VEGFR1 (B), VEGFR2 (C), pEGFR/EGFR (D), pAkt/Akt (E), and NF-κB (F) in highly metastatic LLC cells The cells were treated for 48 h with the indicated concentrations of TFC and TNC (TFC16/TNC16–TFC250/TNC250: 0.016–0.25 mM), Ly (0.016 mM) and Bay (0.0032 mM). The protein expressions were analyzed by Western Blotting. The optical density (OD) of the band is normalized with respective β-actin and is expressed as relative optical density (OD). The OD value of the band shown as mean ± SD is relative to that of the control (DMSO vehicle) designated as 100%. Bay and Ly are the inhibitors of NF- κ B and PI3K/Akt, respectively. For one experiment, 3 assays were carried out and only one set of gels is shown. Values with different letters (a–g) differ significantly ( P

    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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    <t>EREG</t> binds to <t>EGFR</t> via N57 and requires the EGFR domains I and III. (A) HEK293 cells were transiently coexpressed with FLAG-EREG and HA-tagged EGFR. Cell extracts were immunoprecipitated separately with anti-FLAG or anti-HA antibodies, and the associated EGFR and EREG proteins were examined by Western blotting. (B) Endogenous EGFR and EREG were immunoprecipitated from HN4 cells, and bound endogenous EREG and EGFR were examined by Western blotting. (C) The cellular location of EGFR (red) and EREG (green) was examined by immunofluorescence staining (nuclei were stained with DAPI; blue). Scale bar, 50 μm. (D) Schematic diagram of the WT, domain I deletion (∆D1), and domain III mutation (D355T/F357A) constructs of EGFR (FLAG-EGFR-ECD). The numbers represent amino acid residues. (E-F) FLAG-tagged WT or deletion mutants of EGFR were coexpressed with HA-EREG in HEK293 cells. Extracts were immunoprecipitated with an anti-FLAG or anti-HA antibody, and bound EREG or EGFR was examined by Western blotting using the anti-HA or anti-FLAG antibody (for input control, see Figure S4C ). (G) Sequence alignment of EREG from different species. (H) Schematic diagram of various EREG NQ mutants used in this study. The numbers indicate amino acid positions on the EREG. (I-J) HA-tagged WT or NQ mutants of EREG were coexpressed with FLAG-EGFR in HEK293 cells. EREG and EGFR were immunoprecipitated with anti-HA and anti-FLAG antibodies, respectively, and analyzed by Western blotting.
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    EREG binds to EGFR via N57 and requires the EGFR domains I and III. (A) HEK293 cells were transiently coexpressed with FLAG-EREG and HA-tagged EGFR. Cell extracts were immunoprecipitated separately with anti-FLAG or anti-HA antibodies, and the associated EGFR and EREG proteins were examined by Western blotting. (B) Endogenous EGFR and EREG were immunoprecipitated from HN4 cells, and bound endogenous EREG and EGFR were examined by Western blotting. (C) The cellular location of EGFR (red) and EREG (green) was examined by immunofluorescence staining (nuclei were stained with DAPI; blue). Scale bar, 50 μm. (D) Schematic diagram of the WT, domain I deletion (∆D1), and domain III mutation (D355T/F357A) constructs of EGFR (FLAG-EGFR-ECD). The numbers represent amino acid residues. (E-F) FLAG-tagged WT or deletion mutants of EGFR were coexpressed with HA-EREG in HEK293 cells. Extracts were immunoprecipitated with an anti-FLAG or anti-HA antibody, and bound EREG or EGFR was examined by Western blotting using the anti-HA or anti-FLAG antibody (for input control, see Figure S4C ). (G) Sequence alignment of EREG from different species. (H) Schematic diagram of various EREG NQ mutants used in this study. The numbers indicate amino acid positions on the EREG. (I-J) HA-tagged WT or NQ mutants of EREG were coexpressed with FLAG-EGFR in HEK293 cells. EREG and EGFR were immunoprecipitated with anti-HA and anti-FLAG antibodies, respectively, and analyzed by Western blotting.

    Journal: Theranostics

    Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

    doi: 10.7150/thno.47176

    Figure Lengend Snippet: EREG binds to EGFR via N57 and requires the EGFR domains I and III. (A) HEK293 cells were transiently coexpressed with FLAG-EREG and HA-tagged EGFR. Cell extracts were immunoprecipitated separately with anti-FLAG or anti-HA antibodies, and the associated EGFR and EREG proteins were examined by Western blotting. (B) Endogenous EGFR and EREG were immunoprecipitated from HN4 cells, and bound endogenous EREG and EGFR were examined by Western blotting. (C) The cellular location of EGFR (red) and EREG (green) was examined by immunofluorescence staining (nuclei were stained with DAPI; blue). Scale bar, 50 μm. (D) Schematic diagram of the WT, domain I deletion (∆D1), and domain III mutation (D355T/F357A) constructs of EGFR (FLAG-EGFR-ECD). The numbers represent amino acid residues. (E-F) FLAG-tagged WT or deletion mutants of EGFR were coexpressed with HA-EREG in HEK293 cells. Extracts were immunoprecipitated with an anti-FLAG or anti-HA antibody, and bound EREG or EGFR was examined by Western blotting using the anti-HA or anti-FLAG antibody (for input control, see Figure S4C ). (G) Sequence alignment of EREG from different species. (H) Schematic diagram of various EREG NQ mutants used in this study. The numbers indicate amino acid positions on the EREG. (I-J) HA-tagged WT or NQ mutants of EREG were coexpressed with FLAG-EGFR in HEK293 cells. EREG and EGFR were immunoprecipitated with anti-HA and anti-FLAG antibodies, respectively, and analyzed by Western blotting.

    Article Snippet: Antibodies against EREG, EGFR, p-EGFR, ERK, p-ERK, AKT, p-AKT, ErbB2, p-ErbB2, ErbB3, p-ErbB3, STAT3, p-SATAT3, C-Myc, Axl and p-Axl were purchased from Cell Signaling Technology (MA, USA).

    Techniques: Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Mutagenesis, Construct, Sequencing

    EGFR-Erk activation by epiregulin is sustained. (A) Representative time courses of EGFR phosphorylation at Y1086 in SACC-83 cells induced by EGF, EREG, AREG or TGF-α. An anti-EGFR antibody was used as a loading control. (B) Representative time courses of EGFR phosphorylation at Y1173 in CAL27 cells induced by EGF, EREG, AREG or TGF-α. An anti-EGFR antibody was used as a loading control. (C-D) Quantification of EGFR phosphorylation time courses, normalized by the signal at 5 min. The data are plotted on the same graph for multiple independent experiments quantitating phosphorylation at Y1068 and Y1173. (E-F) Representative time courses of Erk phosphorylation in SACC-83 and CAL27 cells induced by different EGFR ligands. (G-H) Quantification of Erk phosphorylation time courses, normalized by the signal at 5 min. The data are plotted on the same graph for multiple independent experiments quantifying Erk phosphorylation.

    Journal: Theranostics

    Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

    doi: 10.7150/thno.47176

    Figure Lengend Snippet: EGFR-Erk activation by epiregulin is sustained. (A) Representative time courses of EGFR phosphorylation at Y1086 in SACC-83 cells induced by EGF, EREG, AREG or TGF-α. An anti-EGFR antibody was used as a loading control. (B) Representative time courses of EGFR phosphorylation at Y1173 in CAL27 cells induced by EGF, EREG, AREG or TGF-α. An anti-EGFR antibody was used as a loading control. (C-D) Quantification of EGFR phosphorylation time courses, normalized by the signal at 5 min. The data are plotted on the same graph for multiple independent experiments quantitating phosphorylation at Y1068 and Y1173. (E-F) Representative time courses of Erk phosphorylation in SACC-83 and CAL27 cells induced by different EGFR ligands. (G-H) Quantification of Erk phosphorylation time courses, normalized by the signal at 5 min. The data are plotted on the same graph for multiple independent experiments quantifying Erk phosphorylation.

    Article Snippet: Antibodies against EREG, EGFR, p-EGFR, ERK, p-ERK, AKT, p-AKT, ErbB2, p-ErbB2, ErbB3, p-ErbB3, STAT3, p-SATAT3, C-Myc, Axl and p-Axl were purchased from Cell Signaling Technology (MA, USA).

    Techniques: Activation Assay

    EREG-induced C-Myc expression depends on EGFR activity. (A) Western blot analysis of C-Myc, p-EGFR, and EGFR from tumor cell lines pretreated with various EGFR inhibitors for 1 h followed by stimulation with epiregulin for 2 h. (B) OncoPrint of EREG-EGFR-MYC pathway alterations in HNC. Genomic alterations of different members of this pathway are mutually exclusive. (C) Western blot analysis of C-Myc, p-EGFR, EGFR, p-AKT, AKT, p-ERK, ERK, p-STAT3, and STAT3 from CAL27 and HN6 cells pretreated with various inhibitors for 1 h followed by stimulation with epiregulin for 2 h.

    Journal: Theranostics

    Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

    doi: 10.7150/thno.47176

    Figure Lengend Snippet: EREG-induced C-Myc expression depends on EGFR activity. (A) Western blot analysis of C-Myc, p-EGFR, and EGFR from tumor cell lines pretreated with various EGFR inhibitors for 1 h followed by stimulation with epiregulin for 2 h. (B) OncoPrint of EREG-EGFR-MYC pathway alterations in HNC. Genomic alterations of different members of this pathway are mutually exclusive. (C) Western blot analysis of C-Myc, p-EGFR, EGFR, p-AKT, AKT, p-ERK, ERK, p-STAT3, and STAT3 from CAL27 and HN6 cells pretreated with various inhibitors for 1 h followed by stimulation with epiregulin for 2 h.

    Article Snippet: Antibodies against EREG, EGFR, p-EGFR, ERK, p-ERK, AKT, p-AKT, ErbB2, p-ErbB2, ErbB3, p-ErbB3, STAT3, p-SATAT3, C-Myc, Axl and p-Axl were purchased from Cell Signaling Technology (MA, USA).

    Techniques: Expressing, Activity Assay, Western Blot

    EREG associates with EGFR and triggers EGFR signaling. (A) Immunoblot (IB) of HN13 and HN6 cells treated with epiregulin (50 ng/ml) at the indicated time points and probed with an anti-phosphotyrosine (p-Tyr) antibody. (B) IB of SACC and HNSCC cancer cells treated with epiregulin (50 ng/ml) for 5 min and probed with an anti-p-Tyr antibody. (C) Human phospho-RTK antibody array analysis of HN6 cells serum starved for 24 hr, followed by epiregulin (50 ng/mL) treatment for 5 min. (D) IB of HN13 (left) and HN6 (right) cells treated with epiregulin (50 ng/ml) at various time points. (E) Immunofluorescence staining for EGFR in HN6 cells treated with or without 50 ng/mL epiregulin.

    Journal: Theranostics

    Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

    doi: 10.7150/thno.47176

    Figure Lengend Snippet: EREG associates with EGFR and triggers EGFR signaling. (A) Immunoblot (IB) of HN13 and HN6 cells treated with epiregulin (50 ng/ml) at the indicated time points and probed with an anti-phosphotyrosine (p-Tyr) antibody. (B) IB of SACC and HNSCC cancer cells treated with epiregulin (50 ng/ml) for 5 min and probed with an anti-p-Tyr antibody. (C) Human phospho-RTK antibody array analysis of HN6 cells serum starved for 24 hr, followed by epiregulin (50 ng/mL) treatment for 5 min. (D) IB of HN13 (left) and HN6 (right) cells treated with epiregulin (50 ng/ml) at various time points. (E) Immunofluorescence staining for EGFR in HN6 cells treated with or without 50 ng/mL epiregulin.

    Article Snippet: Antibodies against EREG, EGFR, p-EGFR, ERK, p-ERK, AKT, p-AKT, ErbB2, p-ErbB2, ErbB3, p-ErbB3, STAT3, p-SATAT3, C-Myc, Axl and p-Axl were purchased from Cell Signaling Technology (MA, USA).

    Techniques: Ab Array, Immunofluorescence, Staining

    EREG triggers EGFR downstream signaling in an EGFR kinase-dependent manner. (A) HN13 and HN6 cells pretreated with erlotinib and AG1478 followed by epiregulin treatment and IB with the indicated antibodies. (B) IB of HN6 and HN13 cells transfected with individual small interfering RNAs (siRNAs) against EGFR in the presence or absence of epiregulin (50 ng/ml). (C) IB of HN6 and HN12 cells treated with or without epiregulin (50 ng/ml). (D) Human phosphokinase antibody array analysis of HN6 cells treated with or without epiregulin (50 ng/ml) for 5 min. (E) IB of HN13 (left) and HN6 (right) cells treated with epiregulin (50 ng/ml) at different time points. (F) HN6 and HN13 cells pretreated with erlotinib and AG1478 followed by epiregulin treatment and immunoblotting (IB) with the indicated antibodies.

    Journal: Theranostics

    Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

    doi: 10.7150/thno.47176

    Figure Lengend Snippet: EREG triggers EGFR downstream signaling in an EGFR kinase-dependent manner. (A) HN13 and HN6 cells pretreated with erlotinib and AG1478 followed by epiregulin treatment and IB with the indicated antibodies. (B) IB of HN6 and HN13 cells transfected with individual small interfering RNAs (siRNAs) against EGFR in the presence or absence of epiregulin (50 ng/ml). (C) IB of HN6 and HN12 cells treated with or without epiregulin (50 ng/ml). (D) Human phosphokinase antibody array analysis of HN6 cells treated with or without epiregulin (50 ng/ml) for 5 min. (E) IB of HN13 (left) and HN6 (right) cells treated with epiregulin (50 ng/ml) at different time points. (F) HN6 and HN13 cells pretreated with erlotinib and AG1478 followed by epiregulin treatment and immunoblotting (IB) with the indicated antibodies.

    Article Snippet: Antibodies against EREG, EGFR, p-EGFR, ERK, p-ERK, AKT, p-AKT, ErbB2, p-ErbB2, ErbB3, p-ErbB3, STAT3, p-SATAT3, C-Myc, Axl and p-Axl were purchased from Cell Signaling Technology (MA, USA).

    Techniques: Transfection, Ab Array

    The proposed model showing that EGFR domains I and III and the N57 residue of EREG are required for EREG-induced EGFR-Erk-C-Myc signaling activation, which in turn promotes oncogenesis and increases erlotinib sensitivity in HNSCC patients.

    Journal: Theranostics

    Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

    doi: 10.7150/thno.47176

    Figure Lengend Snippet: The proposed model showing that EGFR domains I and III and the N57 residue of EREG are required for EREG-induced EGFR-Erk-C-Myc signaling activation, which in turn promotes oncogenesis and increases erlotinib sensitivity in HNSCC patients.

    Article Snippet: Antibodies against EREG, EGFR, p-EGFR, ERK, p-ERK, AKT, p-AKT, ErbB2, p-ErbB2, ErbB3, p-ErbB3, STAT3, p-SATAT3, C-Myc, Axl and p-Axl were purchased from Cell Signaling Technology (MA, USA).

    Techniques: Activation Assay

    Upstream EGFR/ErbB2 signaling mediates HSF1 activation in a mutp53-dependent manner. ( a ) Dual inhibition of EGFR/ErbB2 inhibits HSF1 activation. ErbB2+ breast cancer cells SKBr3 were treated with CP724714 (10 μ M for 24 h) or vehicle. ( a – h ) Immunoblot analysis. ( b ) Stimulation of EGFR signaling induces Ser326 HSF1 phosphorylation. MDA231 cells serum-starved overnight and treated with EGF (30 ng/ml) for 10 min before harvesting. ( c ) Overexpression of mutp53 potentiates EGFR signaling after EGF stimulation. Increased phosphorylation of EGFR-Tyr845 and downstream effectors in MDA231R280K vs vector controls. Treatment as in b . ( d ) Stable overexpression of native p53R175H in SKBr3 increases the levels of ErbB2, pAKT and the HSF1 response. ( e ) Depletion of mutp53 or HSF1 in SKBr3 cells reduces both EGFR and p-Ser326 HSF1 levels. Transfection with scrambled, p53 or HSF1 siRNAs. ( f ) Left, the mutp53 allele in MECs from p53H/+ErbB2 mice correlates with increased levels of ErbB2 and higher activity of HSF1, compared with the p53 null allele. Right , even in the absence of the ErbB2 transgene, MECs from p53H/H mice have higher levels of ErbB2 and heat-shock proteins than p53−/− MECs. ( g ) Cell lines, established from primary mammary tumors of H/H;ErbB2 mouse showed elevated levels of ErbB2, EGFR and activated HSF1 (indicated by increased Hsp70) compared with p53−/−ErbB2 littermate control. ( h ) Dual inhibition of PI3K and MAPK signaling decreases EGFR levels in a mutp53-dependent manner. MDA231 vector and MDA231R280K cells were treated with low or high concentrations of UO126 (10 or 20 μ M) and LY294002 (5 or 10 μ M) for 48 h. ( i ) p53 and p-Ser326 HSF1 immunohistochemical staining of tissue microarray of 150 primary invasive human breast cancers from 75 different patients (two biopsies from separate tumor regions per patient). Representative cases are shown. Strong p53 staining was used as surrogate marker for p53 mutation and a nuclear signal with a p-Ser326-specific HSF1 antibody as marker for HSF1 activation. Staining intensities were blindly scored from 0 to 4. A clear correlation between levels of mutp53 stabilization and nuclear p-Ser326 HSF1 were present in strong (3+) Her2-positive tumors (rho=0.213, P =0.008; n =85 cores) but not in Her2 weak or negative tumors (rho=0.243, P =0.932; n =65 cores). See also Supplementary Figure 1 . ( j ) Proposed model. mutp53, by enhancing EGFR/ErbB2/MAPK/PI3K signaling, potentiates HSF1 activity via a feed forward circuit and thereby endows cancer cells with superior tolerance to proteotoxic stress

    Journal: Cell Death & Disease

    Article Title: A gain-of-function mutant p53-HSF1 feed forward circuit governs adaptation of cancer cells to proteotoxic stress

    doi: 10.1038/cddis.2014.158

    Figure Lengend Snippet: Upstream EGFR/ErbB2 signaling mediates HSF1 activation in a mutp53-dependent manner. ( a ) Dual inhibition of EGFR/ErbB2 inhibits HSF1 activation. ErbB2+ breast cancer cells SKBr3 were treated with CP724714 (10 μ M for 24 h) or vehicle. ( a – h ) Immunoblot analysis. ( b ) Stimulation of EGFR signaling induces Ser326 HSF1 phosphorylation. MDA231 cells serum-starved overnight and treated with EGF (30 ng/ml) for 10 min before harvesting. ( c ) Overexpression of mutp53 potentiates EGFR signaling after EGF stimulation. Increased phosphorylation of EGFR-Tyr845 and downstream effectors in MDA231R280K vs vector controls. Treatment as in b . ( d ) Stable overexpression of native p53R175H in SKBr3 increases the levels of ErbB2, pAKT and the HSF1 response. ( e ) Depletion of mutp53 or HSF1 in SKBr3 cells reduces both EGFR and p-Ser326 HSF1 levels. Transfection with scrambled, p53 or HSF1 siRNAs. ( f ) Left, the mutp53 allele in MECs from p53H/+ErbB2 mice correlates with increased levels of ErbB2 and higher activity of HSF1, compared with the p53 null allele. Right , even in the absence of the ErbB2 transgene, MECs from p53H/H mice have higher levels of ErbB2 and heat-shock proteins than p53−/− MECs. ( g ) Cell lines, established from primary mammary tumors of H/H;ErbB2 mouse showed elevated levels of ErbB2, EGFR and activated HSF1 (indicated by increased Hsp70) compared with p53−/−ErbB2 littermate control. ( h ) Dual inhibition of PI3K and MAPK signaling decreases EGFR levels in a mutp53-dependent manner. MDA231 vector and MDA231R280K cells were treated with low or high concentrations of UO126 (10 or 20 μ M) and LY294002 (5 or 10 μ M) for 48 h. ( i ) p53 and p-Ser326 HSF1 immunohistochemical staining of tissue microarray of 150 primary invasive human breast cancers from 75 different patients (two biopsies from separate tumor regions per patient). Representative cases are shown. Strong p53 staining was used as surrogate marker for p53 mutation and a nuclear signal with a p-Ser326-specific HSF1 antibody as marker for HSF1 activation. Staining intensities were blindly scored from 0 to 4. A clear correlation between levels of mutp53 stabilization and nuclear p-Ser326 HSF1 were present in strong (3+) Her2-positive tumors (rho=0.213, P =0.008; n =85 cores) but not in Her2 weak or negative tumors (rho=0.243, P =0.932; n =65 cores). See also Supplementary Figure 1 . ( j ) Proposed model. mutp53, by enhancing EGFR/ErbB2/MAPK/PI3K signaling, potentiates HSF1 activity via a feed forward circuit and thereby endows cancer cells with superior tolerance 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 .

    Techniques: Activation Assay, Inhibition, Over Expression, Plasmid Preparation, Transfection, Mouse Assay, Activity Assay, Immunohistochemistry, Staining, Microarray, Marker, Mutagenesis

    Knockdown of endogenous Giα2 has differential effects on PI3-kinase activation and cell migration in response to diverse ligands A . Western blot analysis of p-AKT and p-EGFR in PC3 cells lysates, transfected with control siRNA or Giα2 siRNA in response to OXT (100 nmol/L), TGFβ1 (5 ng/ml) or EGF (3 ng/ml). Total AKT and EGFR served as loading and normalization controls. B . Quantitative analysis of p-AKT activation in PC3 cells. Data is presented as mean ± SEM (n=3) and analyzed by ANOVA and Duncan’s modified range tests. Significant differences between groups in a given category (P

    Journal: Journal of cellular physiology

    Article Title: Novel role of Giα2 in cell migration: Downstream of PI3-kinase/AKT and Rac1 in prostate cancer cells

    doi: 10.1002/jcp.26894

    Figure Lengend Snippet: Knockdown of endogenous Giα2 has differential effects on PI3-kinase activation and cell migration in response to diverse ligands A . Western blot analysis of p-AKT and p-EGFR in PC3 cells lysates, transfected with control siRNA or Giα2 siRNA in response to OXT (100 nmol/L), TGFβ1 (5 ng/ml) or EGF (3 ng/ml). Total AKT and EGFR served as loading and normalization controls. B . Quantitative analysis of p-AKT activation in PC3 cells. Data is presented as mean ± SEM (n=3) and analyzed by ANOVA and Duncan’s modified range tests. Significant differences between groups in a given category (P

    Article Snippet: Antibodies against phospho-AKTser473 , AKT, phospho-EGFR and EGFR were purchased from Cell Signaling Technology, Inc. (Danvers, MA).

    Techniques: Activation Assay, Migration, Western Blot, Transfection, Modification

    A secreted EPEC component induces EGFR autophosphorylation. (A) C2 BBE cells were treated with filter-sterilized EPEC supernatants for 60, 120, and 240 min. The monolayers were washed and the cell extracts (300 μg) immunoblotted against EGFR pY1068, total EGFR, and actin. (B) Data from three similar experiments were evaluated by densitometry ( P

    Journal: Infection and Immunity

    Article Title: Enteropathogenic Escherichia coli-Induced Epidermal Growth Factor Receptor Activation Contributes to Physiological Alterations in Intestinal Epithelial Cells ▿

    doi: 10.1128/IAI.01690-06

    Figure Lengend Snippet: A secreted EPEC component induces EGFR autophosphorylation. (A) C2 BBE cells were treated with filter-sterilized EPEC supernatants for 60, 120, and 240 min. The monolayers were washed and the cell extracts (300 μg) immunoblotted against EGFR pY1068, total EGFR, and actin. (B) Data from three similar experiments were evaluated by densitometry ( P

    Article Snippet: Antibodies against phosphorylated EGFR pY1068 were obtained from Cell Signaling (Beverly, MA).

    Techniques:

    EPEC transactivates EGFR in intestinal epithelial cells. (A) Cell extracts (300 μg) from C2 BBE cells infected with WT EPEC or the commensal strain HS-4 for 60, 120, and 240 min were immunoblotted against EGFR pY1068, total EGFR, and actin. (B) Densitometric analysis was performed on three such blots to determine pEGFR abundance relative to total EGFR and normalized to levels in control uninfected cells ( P

    Journal: Infection and Immunity

    Article Title: Enteropathogenic Escherichia coli-Induced Epidermal Growth Factor Receptor Activation Contributes to Physiological Alterations in Intestinal Epithelial Cells ▿

    doi: 10.1128/IAI.01690-06

    Figure Lengend Snippet: EPEC transactivates EGFR in intestinal epithelial cells. (A) Cell extracts (300 μg) from C2 BBE cells infected with WT EPEC or the commensal strain HS-4 for 60, 120, and 240 min were immunoblotted against EGFR pY1068, total EGFR, and actin. (B) Densitometric analysis was performed on three such blots to determine pEGFR abundance relative to total EGFR and normalized to levels in control uninfected cells ( P

    Article Snippet: Antibodies against phosphorylated EGFR pY1068 were obtained from Cell Signaling (Beverly, MA).

    Techniques: Infection

    EPEC-induced EGFR activation is delayed in polarized epithelial cells. (A) Polarized C2 BBE cells grown on permeable supports were infected with WT EPEC, and the extracts isolated at specific time points were immunoblotted against EGFR pY1068. EGF-treated A431 cell extract was included as a positive control to confirm the identity of the band. (B) Data from three similar experiments were evaluated by densitometry ( P

    Journal: Infection and Immunity

    Article Title: Enteropathogenic Escherichia coli-Induced Epidermal Growth Factor Receptor Activation Contributes to Physiological Alterations in Intestinal Epithelial Cells ▿

    doi: 10.1128/IAI.01690-06

    Figure Lengend Snippet: EPEC-induced EGFR activation is delayed in polarized epithelial cells. (A) Polarized C2 BBE cells grown on permeable supports were infected with WT EPEC, and the extracts isolated at specific time points were immunoblotted against EGFR pY1068. EGF-treated A431 cell extract was included as a positive control to confirm the identity of the band. (B) Data from three similar experiments were evaluated by densitometry ( P

    Article Snippet: Antibodies against phosphorylated EGFR pY1068 were obtained from Cell Signaling (Beverly, MA).

    Techniques: Activation Assay, Infection, Isolation, Positive Control