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  • 97
    Name:
    EGFR
    Description:
    supplied as 0 5 mg 0 1 ml substrate suitable for kinase assays Suitable substrate EGFR sc 4819 Store at 20° C Recognition Motif E D Y X
    Catalog Number:
    SC-24615
    Price:
    None
    Category:
    Chemicals Other Chemicals Other Substrates EGFR Thr 1173
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    Structured Review

    Santa Cruz Biotechnology egfr
    dmp binding to IR augments insulin signalling pathway. (A) dmp fails to activate <t>EGFR.</t> L6 myotubes or 3T3L1 adipocytes were treated with or without 250 nM dmp for 4h. The cell lysates were analyzed by immunoblotting with <t>anti-pEGFR</t> and anti-EGFR antibodies. (B,C) dmp can induce IR phosphorylation in a dose dependent manner. L6 myotubes were treated with insulin (20–120 n m ) or dmp (50–300 n m ) for 4h and IR phosphorylation was monitored by ELISA (B) or immunoblotting with anti-pIR and anti-IR antibodies (C). (D) IR kinase activity was determined in L6 myotubes which were incubated with varied concentrations of insulin or dmp. (E) dmp stimulates IR and its downstream kinases phosphorylation. L6 myotubes or 3T3L1 adipocytes were treated with or without Insulin (100 nM) or dmp (250 nM) for 4h and the IR phosphorylation and its downstream signalling were monitored by immunoblotting. (F) L6 myotubes transfected with GFP-GLUT4 chimeric gene were incubated with insulin (100 nM) or dmp (250nM) for 4h. Cells on the cover slips were fixed in paraformaldehyde and observed under florescent microscope for GFP-GLUT4 translocation. (G) dmp like insulin promotes glucose uptake. L6 myotubes or skeletal muscle cells from soleus muscle of neonatal mice (2-3days) were incubated with 100 n m insulin or 250 n m dmp for 25 min. [ 14 C] 2-DOG was then added, and the cells were further incubated for 5 min. [ 14 C] 2-DOG uptake was measured by scintillation counting. * P
    supplied as 0 5 mg 0 1 ml substrate suitable for kinase assays Suitable substrate EGFR sc 4819 Store at 20° C Recognition Motif E D Y X
    https://www.bioz.com/result/egfr/product/Santa Cruz Biotechnology
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    egfr - by Bioz Stars, 2021-05
    97/100 stars

    Images

    1) Product Images from "A Small Insulinomimetic Molecule Also Improves Insulin Sensitivity in Diabetic Mice"

    Article Title: A Small Insulinomimetic Molecule Also Improves Insulin Sensitivity in Diabetic Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0169809

    dmp binding to IR augments insulin signalling pathway. (A) dmp fails to activate EGFR. L6 myotubes or 3T3L1 adipocytes were treated with or without 250 nM dmp for 4h. The cell lysates were analyzed by immunoblotting with anti-pEGFR and anti-EGFR antibodies. (B,C) dmp can induce IR phosphorylation in a dose dependent manner. L6 myotubes were treated with insulin (20–120 n m ) or dmp (50–300 n m ) for 4h and IR phosphorylation was monitored by ELISA (B) or immunoblotting with anti-pIR and anti-IR antibodies (C). (D) IR kinase activity was determined in L6 myotubes which were incubated with varied concentrations of insulin or dmp. (E) dmp stimulates IR and its downstream kinases phosphorylation. L6 myotubes or 3T3L1 adipocytes were treated with or without Insulin (100 nM) or dmp (250 nM) for 4h and the IR phosphorylation and its downstream signalling were monitored by immunoblotting. (F) L6 myotubes transfected with GFP-GLUT4 chimeric gene were incubated with insulin (100 nM) or dmp (250nM) for 4h. Cells on the cover slips were fixed in paraformaldehyde and observed under florescent microscope for GFP-GLUT4 translocation. (G) dmp like insulin promotes glucose uptake. L6 myotubes or skeletal muscle cells from soleus muscle of neonatal mice (2-3days) were incubated with 100 n m insulin or 250 n m dmp for 25 min. [ 14 C] 2-DOG was then added, and the cells were further incubated for 5 min. [ 14 C] 2-DOG uptake was measured by scintillation counting. * P
    Figure Legend Snippet: dmp binding to IR augments insulin signalling pathway. (A) dmp fails to activate EGFR. L6 myotubes or 3T3L1 adipocytes were treated with or without 250 nM dmp for 4h. The cell lysates were analyzed by immunoblotting with anti-pEGFR and anti-EGFR antibodies. (B,C) dmp can induce IR phosphorylation in a dose dependent manner. L6 myotubes were treated with insulin (20–120 n m ) or dmp (50–300 n m ) for 4h and IR phosphorylation was monitored by ELISA (B) or immunoblotting with anti-pIR and anti-IR antibodies (C). (D) IR kinase activity was determined in L6 myotubes which were incubated with varied concentrations of insulin or dmp. (E) dmp stimulates IR and its downstream kinases phosphorylation. L6 myotubes or 3T3L1 adipocytes were treated with or without Insulin (100 nM) or dmp (250 nM) for 4h and the IR phosphorylation and its downstream signalling were monitored by immunoblotting. (F) L6 myotubes transfected with GFP-GLUT4 chimeric gene were incubated with insulin (100 nM) or dmp (250nM) for 4h. Cells on the cover slips were fixed in paraformaldehyde and observed under florescent microscope for GFP-GLUT4 translocation. (G) dmp like insulin promotes glucose uptake. L6 myotubes or skeletal muscle cells from soleus muscle of neonatal mice (2-3days) were incubated with 100 n m insulin or 250 n m dmp for 25 min. [ 14 C] 2-DOG was then added, and the cells were further incubated for 5 min. [ 14 C] 2-DOG uptake was measured by scintillation counting. * P

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Incubation, Transfection, Microscopy, Translocation Assay, Mouse Assay

    2) Product Images from "The Influence of TGF-β3, EGF, and BGN on SOX9 and RUNX2 Expression in Human Chondrogenic Progenitor Cells"

    Article Title: The Influence of TGF-β3, EGF, and BGN on SOX9 and RUNX2 Expression in Human Chondrogenic Progenitor Cells

    Journal: Journal of Histochemistry and Cytochemistry

    doi: 10.1369/0022155418811645

    Stimulation and knockdown experiments affect CPCs. (A) Effect of TGF-β3 and EGF stimulation on the TGFBRI, SMAD2, EGFR, and BGN mRNA level. (B). Representative WB of p-SMAD2 after TGF-β3 and EGF stimulation. (C) Effect of BGN knockdown on EGFR protein level. (D) Representative WB of BGN knockdown. (E) Stimulation of CPCs by TGF-β3 and EGF altered SOX9 and RUNX2 mRNA expression. (F) Analysis of SOX9 protein level after stimulation with EGF. (G) Representative WB of SOX9 after EGF stimulation. (H) Chondrogenic and (I) osteogenic (COL1) as well as hypertrophy (COL10) marker expression after TGF-β3 and EGF stimulation. Abbreviations: ACAN, aggrecan; COL, collagen; TGFBRI, TGF-beta receptor type-1; TGF-β3, transforming growth factor beta-3; EGFR, epidermal growth factor receptor; BGN, biglycan; EGF, epidermal growth factor; SMAD2, mothers against decapentaplegic homolog 2; WB, Western blotting; CPC, chondrogenic progenitor cell.
    Figure Legend Snippet: Stimulation and knockdown experiments affect CPCs. (A) Effect of TGF-β3 and EGF stimulation on the TGFBRI, SMAD2, EGFR, and BGN mRNA level. (B). Representative WB of p-SMAD2 after TGF-β3 and EGF stimulation. (C) Effect of BGN knockdown on EGFR protein level. (D) Representative WB of BGN knockdown. (E) Stimulation of CPCs by TGF-β3 and EGF altered SOX9 and RUNX2 mRNA expression. (F) Analysis of SOX9 protein level after stimulation with EGF. (G) Representative WB of SOX9 after EGF stimulation. (H) Chondrogenic and (I) osteogenic (COL1) as well as hypertrophy (COL10) marker expression after TGF-β3 and EGF stimulation. Abbreviations: ACAN, aggrecan; COL, collagen; TGFBRI, TGF-beta receptor type-1; TGF-β3, transforming growth factor beta-3; EGFR, epidermal growth factor receptor; BGN, biglycan; EGF, epidermal growth factor; SMAD2, mothers against decapentaplegic homolog 2; WB, Western blotting; CPC, chondrogenic progenitor cell.

    Techniques Used: Western Blot, Expressing, Marker

    CPCs were stained for TGFBRI and EGFR by ICC. (A and E) Nuclei were stained with DAPI. (B and F) Staining of BGN. (C) TGFBRI signal. (D) Colocalization of BGN and TGFBRI. (G) EGFR signal. (H) Colocalization of BGN and EGFR. Scale bar: 75 µm. Abbreviations: CPCs, chondrogenic progenitor cells; TGFBRI, TGF-beta receptor type-1; EGFR, epidermal growth factor receptor; ICC, immunocytochemistry; DAPI, 4′,6-diamidino-2-phenylindole; BGN, biglycan.
    Figure Legend Snippet: CPCs were stained for TGFBRI and EGFR by ICC. (A and E) Nuclei were stained with DAPI. (B and F) Staining of BGN. (C) TGFBRI signal. (D) Colocalization of BGN and TGFBRI. (G) EGFR signal. (H) Colocalization of BGN and EGFR. Scale bar: 75 µm. Abbreviations: CPCs, chondrogenic progenitor cells; TGFBRI, TGF-beta receptor type-1; EGFR, epidermal growth factor receptor; ICC, immunocytochemistry; DAPI, 4′,6-diamidino-2-phenylindole; BGN, biglycan.

    Techniques Used: Staining, Immunocytochemistry

    Detection of BGN, TGFBRI, and EGFR. Staining of BGN in (A) healthy tissue and (B) late-stage OA tissue. CC-D exhibited positive IHC staining of TGFBRI in the (C) middle and (D) deep zones. (E) Membranous staining of TGFBRI in CC-D. EGFR staining of CC-D residing in the middle (F) and deep (G) zones. (H) Membranous staining of EGFR in CC-D. Relative expression levels of (I) TGFBRI and (J) EGFR mRNA in CC-D and CPCs compared with in CC-H. Arrows indicate duplication of the tidemark. Scale bar in A and B: 750 µm. Scale bar in C, D, F, and G: 150 µm. Scale bar in E and H: 75 µm. Abbreviations: BGN, biglycan; TGFBRI, TGF-beta receptor type-1; EGFR, epidermal growth factor receptor; OA, osteoarthritis; CC-D, chondrocytes residing in diseased tissue; IHC, immunohistochemistry; CC-H, chondrocytes residing in healthy tissue; CPC, chondrogenic progenitor cell; SZ, superficial zone; MZ, middle zone; DZ, deep zone.
    Figure Legend Snippet: Detection of BGN, TGFBRI, and EGFR. Staining of BGN in (A) healthy tissue and (B) late-stage OA tissue. CC-D exhibited positive IHC staining of TGFBRI in the (C) middle and (D) deep zones. (E) Membranous staining of TGFBRI in CC-D. EGFR staining of CC-D residing in the middle (F) and deep (G) zones. (H) Membranous staining of EGFR in CC-D. Relative expression levels of (I) TGFBRI and (J) EGFR mRNA in CC-D and CPCs compared with in CC-H. Arrows indicate duplication of the tidemark. Scale bar in A and B: 750 µm. Scale bar in C, D, F, and G: 150 µm. Scale bar in E and H: 75 µm. Abbreviations: BGN, biglycan; TGFBRI, TGF-beta receptor type-1; EGFR, epidermal growth factor receptor; OA, osteoarthritis; CC-D, chondrocytes residing in diseased tissue; IHC, immunohistochemistry; CC-H, chondrocytes residing in healthy tissue; CPC, chondrogenic progenitor cell; SZ, superficial zone; MZ, middle zone; DZ, deep zone.

    Techniques Used: Staining, Immunohistochemistry, Expressing

    3) Product Images from "Up-regulation of the kinase gene SGK1 by progesterone activates the AP-1–NDRG1 axis in both PR-positive and -negative breast cancer cells"

    Article Title: Up-regulation of the kinase gene SGK1 by progesterone activates the AP-1–NDRG1 axis in both PR-positive and -negative breast cancer cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.002894

    Depletion of NDRG1 activates multiple cellular kinases and increases migration of MDA-MB-231 cells. A , Western blot analysis depicting knockdown of NDRG1 in MD-231 breast cancer cells (PR-negative). sh-NT was used as vector control for NDRG1 expression. β-Actin protein was used as a loading control for Western blotting. Numbers on the blot indicate the intensity ratio for NDRG1, normalized to respective β-actin levels. The analysis is representative of three independent experiments. B , Western blot analysis of p-EGFR (Tyr-1086), p-AKT (Ser-473), and p-ERK1/2 (Thr-202/Tyr-204) in NDRG1 knockdown clones of MD231 cells. β-Actin was used as a loading control for Western blotting, and β-actin for the p-EGFR and p-ERK1/2 panels is the same. The numbers on the blot indicate average intensity ratio calculated from all of the three replicate experiments for phosphorylation levels of EGFR, AKT, and ERK1/2, normalized to respective total protein levels (EGFR, AKT, and ERK2). Western blot analysis is representative of three independent experiments. C , migration of cells was measured from 0 to 20 h by using a time-lapse wound healing assay. The bar plot represents percentage wound closure (average of the three biological replicate experiments), and the comparison was with respect to sh-NT. The figure is representative of three independent experiments performed in triplicates. p value was calculated using Student's unpaired t test. **, p
    Figure Legend Snippet: Depletion of NDRG1 activates multiple cellular kinases and increases migration of MDA-MB-231 cells. A , Western blot analysis depicting knockdown of NDRG1 in MD-231 breast cancer cells (PR-negative). sh-NT was used as vector control for NDRG1 expression. β-Actin protein was used as a loading control for Western blotting. Numbers on the blot indicate the intensity ratio for NDRG1, normalized to respective β-actin levels. The analysis is representative of three independent experiments. B , Western blot analysis of p-EGFR (Tyr-1086), p-AKT (Ser-473), and p-ERK1/2 (Thr-202/Tyr-204) in NDRG1 knockdown clones of MD231 cells. β-Actin was used as a loading control for Western blotting, and β-actin for the p-EGFR and p-ERK1/2 panels is the same. The numbers on the blot indicate average intensity ratio calculated from all of the three replicate experiments for phosphorylation levels of EGFR, AKT, and ERK1/2, normalized to respective total protein levels (EGFR, AKT, and ERK2). Western blot analysis is representative of three independent experiments. C , migration of cells was measured from 0 to 20 h by using a time-lapse wound healing assay. The bar plot represents percentage wound closure (average of the three biological replicate experiments), and the comparison was with respect to sh-NT. The figure is representative of three independent experiments performed in triplicates. p value was calculated using Student's unpaired t test. **, p

    Techniques Used: Migration, Multiple Displacement Amplification, Western Blot, Plasmid Preparation, Expressing, Clone Assay, Wound Healing Assay

    NDRG1 regulates the activation of multiple cellular kinases and cell migration in T47D cells. A , Western blot analysis depicting knockdown of NDRG1 in T47D cells (PR-positive). sh-NT was used as vector control for NDRG1 expression. β-Actin protein was used as a loading control for Western blotting. Numbers on the blot indicate intensity ratio for NDRG1 expression normalized to respective β-actin levels. The analysis is representative of three independent experiments. B , Western blot analysis of p-EGFR (Tyr-1086), p-AKT (Ser-473), and p-ERK1/2 (Thr-202/Tyr-204) in NDRG1 knockdown clones of T47D cells. β-Actin used as a loading control for Western blotting. The figure is representative of three independent experiments. Numbers on the blot indicate average intensity ratio calculated from all of the three replicate experiments for phosphorylation levels of EGFR, AKT, and ERK1/2, normalized to the respective total protein levels (EGFR, AKT, and ERK2). Western blot analysis is representative of three independent experiments. C , cell migration analysis upon depletion of NDRG1 in T47D breast cancer cells. Cells were monitored by a time-lapse wound healing assay for 20 h. Cell migration from the initial to the 20-h time point was plotted as percentage wound closure (average of the three biological replicate experiments), and the comparison was with respect to sh-NT. The figure is representative of three independent experiments performed in triplicates. p value was calculated using Student's unpaired t test. ***, p
    Figure Legend Snippet: NDRG1 regulates the activation of multiple cellular kinases and cell migration in T47D cells. A , Western blot analysis depicting knockdown of NDRG1 in T47D cells (PR-positive). sh-NT was used as vector control for NDRG1 expression. β-Actin protein was used as a loading control for Western blotting. Numbers on the blot indicate intensity ratio for NDRG1 expression normalized to respective β-actin levels. The analysis is representative of three independent experiments. B , Western blot analysis of p-EGFR (Tyr-1086), p-AKT (Ser-473), and p-ERK1/2 (Thr-202/Tyr-204) in NDRG1 knockdown clones of T47D cells. β-Actin used as a loading control for Western blotting. The figure is representative of three independent experiments. Numbers on the blot indicate average intensity ratio calculated from all of the three replicate experiments for phosphorylation levels of EGFR, AKT, and ERK1/2, normalized to the respective total protein levels (EGFR, AKT, and ERK2). Western blot analysis is representative of three independent experiments. C , cell migration analysis upon depletion of NDRG1 in T47D breast cancer cells. Cells were monitored by a time-lapse wound healing assay for 20 h. Cell migration from the initial to the 20-h time point was plotted as percentage wound closure (average of the three biological replicate experiments), and the comparison was with respect to sh-NT. The figure is representative of three independent experiments performed in triplicates. p value was calculated using Student's unpaired t test. ***, p

    Techniques Used: Activation Assay, Migration, Western Blot, Plasmid Preparation, Expressing, Clone Assay, Wound Healing Assay

    4) Product Images from "Combination of EGFR Inhibitor Lapatinib and MET Inhibitor Foretinib Inhibits Migration of Triple Negative Breast Cancer Cell Lines"

    Article Title: Combination of EGFR Inhibitor Lapatinib and MET Inhibitor Foretinib Inhibits Migration of Triple Negative Breast Cancer Cell Lines

    Journal: Cancers

    doi: 10.3390/cancers10090335

    Activation of EGFR, MET, AKT, and ERK in inhibitor-treated TNBC cell lines. Representative immunoblots showing EGFR/pEGFR, MET/pMET, AKT/pAKT, and ERK/pERK levels in cellular extracts of control cells (incubated for 4 h only with 5 nM EGF and 30 ng/mL HGF) and cells treated with HGF, EGF, and the indicated concentrations of foretinib, lapatinib, or their combination. Graphs present densitometric analysis of protein bands for pEGFR, pMET, pERK, and pAKT. ADU stands for arbitrary densitometry units. The densitometry analysis for selected proteins was adjusted using the total protein content. The statistical significance was assessed versus the control. The significance level was set at p ≤ 0.05 (*), p ≤ 0.01 (**), or p ≤ 0.001 (***).
    Figure Legend Snippet: Activation of EGFR, MET, AKT, and ERK in inhibitor-treated TNBC cell lines. Representative immunoblots showing EGFR/pEGFR, MET/pMET, AKT/pAKT, and ERK/pERK levels in cellular extracts of control cells (incubated for 4 h only with 5 nM EGF and 30 ng/mL HGF) and cells treated with HGF, EGF, and the indicated concentrations of foretinib, lapatinib, or their combination. Graphs present densitometric analysis of protein bands for pEGFR, pMET, pERK, and pAKT. ADU stands for arbitrary densitometry units. The densitometry analysis for selected proteins was adjusted using the total protein content. The statistical significance was assessed versus the control. The significance level was set at p ≤ 0.05 (*), p ≤ 0.01 (**), or p ≤ 0.001 (***).

    Techniques Used: Activation Assay, Western Blot, Incubation

    Influence of EGFR and MET inhibitors on the proteolytic activity of the ( A ) MDA-MB-231 and ( B ) BT549 cell lines. Cells were seeded on FITC-gelatin and after inhibitors treatment (12 h) they were fixed and stained for F-actin.Matrix degradation activity was identified by dark areas on the gelatin-FITC background. White arrows indicate invadopodia. Scale bar: 10 µM. The areas digested by ( C ) MDA-MB-231 and ( D ) BT549 cells were calculated using ImageJ software. Asterisks indicate differences between control and treated cells or between cells treated with different drugs. The significance level was set at p ≤ 0.05 (*), p ≤ 0.01 (**), or p ≤ 0.001 (***).
    Figure Legend Snippet: Influence of EGFR and MET inhibitors on the proteolytic activity of the ( A ) MDA-MB-231 and ( B ) BT549 cell lines. Cells were seeded on FITC-gelatin and after inhibitors treatment (12 h) they were fixed and stained for F-actin.Matrix degradation activity was identified by dark areas on the gelatin-FITC background. White arrows indicate invadopodia. Scale bar: 10 µM. The areas digested by ( C ) MDA-MB-231 and ( D ) BT549 cells were calculated using ImageJ software. Asterisks indicate differences between control and treated cells or between cells treated with different drugs. The significance level was set at p ≤ 0.05 (*), p ≤ 0.01 (**), or p ≤ 0.001 (***).

    Techniques Used: Activity Assay, Multiple Displacement Amplification, Staining, Software

    Migration and invasion capacities and invasion velocity of TNBC cells treated with MET and EGFR inhibitors. ( A , C , E ) MDA-MB-231 and ( B , D , F ) BT549 cells were incubated with foretinib (F) lapatinib (L), or their combination at the indicated concentrations (µM) for 24 h or 48 h. ( A , B ) Migration and ( C , D ) invasion experiments were conducted on Transwell filters, non-coated (migration), or coated with collagen type I (invasion). Results are expressed as the mean ± SD and are based on at least three independent experiments. Migration and invasion capacity in control cells is set to 100%. ( E , F ) Invasion velocity of control and treated cells. The change in area of the cell layer was continuously measured and the mean velocity derived from the area increase in time and was calculated ( n = 8). The error bars represent SD. For all graphs, asterisks indicate conditions statistically significant, which is different from the control. The significance level was set at p ≤ 0.05 (*), p ≤ 0.01 (**), or p ≤ 0.001 (***).
    Figure Legend Snippet: Migration and invasion capacities and invasion velocity of TNBC cells treated with MET and EGFR inhibitors. ( A , C , E ) MDA-MB-231 and ( B , D , F ) BT549 cells were incubated with foretinib (F) lapatinib (L), or their combination at the indicated concentrations (µM) for 24 h or 48 h. ( A , B ) Migration and ( C , D ) invasion experiments were conducted on Transwell filters, non-coated (migration), or coated with collagen type I (invasion). Results are expressed as the mean ± SD and are based on at least three independent experiments. Migration and invasion capacity in control cells is set to 100%. ( E , F ) Invasion velocity of control and treated cells. The change in area of the cell layer was continuously measured and the mean velocity derived from the area increase in time and was calculated ( n = 8). The error bars represent SD. For all graphs, asterisks indicate conditions statistically significant, which is different from the control. The significance level was set at p ≤ 0.05 (*), p ≤ 0.01 (**), or p ≤ 0.001 (***).

    Techniques Used: Migration, Multiple Displacement Amplification, Incubation, Derivative Assay

    5) Product Images from "Dasatinib + Gefitinib, a non platinum-based combination with enhanced growth inhibitory, anti-migratory and anti-invasive potency against human ovarian cancer cells"

    Article Title: Dasatinib + Gefitinib, a non platinum-based combination with enhanced growth inhibitory, anti-migratory and anti-invasive potency against human ovarian cancer cells

    Journal: Journal of Ovarian Research

    doi: 10.1186/s13048-017-0319-2

    In vitro molecular effects of Dasatinib, Gefitinib or a combination of both drugs in HOAC signaling pathways. HOAC were treated 2 h or 24 h with Dasatinib (Da), Gefitinib (Ge) (0.1 × IC50 of each cell line after 72 h of treatment) or an equieffective combination of Dasatinib and Gefitinib (Da + Ge) (0.1 × IC50 of each drug alone). The EGFR, phospho-EGFR, c-Src, phospho-c-Src, ERK, phospho-ERK, Akt, phospho-Akt (p-Akt) and β-actin levels were determined by western blot using specific antibodies. Immunocomplexes were visualized by autoradiography
    Figure Legend Snippet: In vitro molecular effects of Dasatinib, Gefitinib or a combination of both drugs in HOAC signaling pathways. HOAC were treated 2 h or 24 h with Dasatinib (Da), Gefitinib (Ge) (0.1 × IC50 of each cell line after 72 h of treatment) or an equieffective combination of Dasatinib and Gefitinib (Da + Ge) (0.1 × IC50 of each drug alone). The EGFR, phospho-EGFR, c-Src, phospho-c-Src, ERK, phospho-ERK, Akt, phospho-Akt (p-Akt) and β-actin levels were determined by western blot using specific antibodies. Immunocomplexes were visualized by autoradiography

    Techniques Used: In Vitro, Western Blot, Autoradiography

    6) Product Images from "Perifosine as a Potential Novel Anti-Cancer Agent Inhibits EGFR/MET-AKT Axis in Malignant Pleural Mesothelioma"

    Article Title: Perifosine as a Potential Novel Anti-Cancer Agent Inhibits EGFR/MET-AKT Axis in Malignant Pleural Mesothelioma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036856

    Effects of perifosine on EGFR and MET phosphorylation and signalling. A, B, Exponentially growing REN cells were pre-treated with 23 µM perifosine for 1 hour and then incubated 10 minutes with EGF (5 ng/ml) or HGF (5 ng/ml). At the end of experiment, cell lysates were prepared or immunoprecipitated and analyzed by immunoblotting with indicated antibodies. Results are representative of three different experiments. B, D, The effect of perifosine was also assessed on growth factor mediated cell proliferation. REN cells were treated with 23 µM perifosine 24 hours in the absence or in the presence of 5 ng/ml EGF or HGF. At the end of treatment viable cell count was determined. Points , means ± SD of three individual measurements.
    Figure Legend Snippet: Effects of perifosine on EGFR and MET phosphorylation and signalling. A, B, Exponentially growing REN cells were pre-treated with 23 µM perifosine for 1 hour and then incubated 10 minutes with EGF (5 ng/ml) or HGF (5 ng/ml). At the end of experiment, cell lysates were prepared or immunoprecipitated and analyzed by immunoblotting with indicated antibodies. Results are representative of three different experiments. B, D, The effect of perifosine was also assessed on growth factor mediated cell proliferation. REN cells were treated with 23 µM perifosine 24 hours in the absence or in the presence of 5 ng/ml EGF or HGF. At the end of treatment viable cell count was determined. Points , means ± SD of three individual measurements.

    Techniques Used: Incubation, Immunoprecipitation, Cell Counting

    7) Product Images from "Antagonism of the prostaglandin E2 EP1 receptor in MDCK cells increases growth through activation of Akt and the epidermal growth factor receptor"

    Article Title: Antagonism of the prostaglandin E2 EP1 receptor in MDCK cells increases growth through activation of Akt and the epidermal growth factor receptor

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00510.2013

    Effect of Akt inhibitor MK-2206 and EGFR inhibitor AG1478 on the growth response to EGFR antagonists. A : effect of 2 μM SC51089 and 70 nM PGE 2 on growth, relative to untreated MDCK cell cultures, was examined both in the presence of 0.5 μM
    Figure Legend Snippet: Effect of Akt inhibitor MK-2206 and EGFR inhibitor AG1478 on the growth response to EGFR antagonists. A : effect of 2 μM SC51089 and 70 nM PGE 2 on growth, relative to untreated MDCK cell cultures, was examined both in the presence of 0.5 μM

    Techniques Used:

    8) Product Images from "Vascular ADAM17 as a Novel Therapeutic Target in Mediating Cardiovascular Hypertrophy and Perivascular Fibrosis Induced by Angiotensin II"

    Article Title: Vascular ADAM17 as a Novel Therapeutic Target in Mediating Cardiovascular Hypertrophy and Perivascular Fibrosis Induced by Angiotensin II

    Journal: Hypertension (Dallas, Tex. : 1979)

    doi: 10.1161/HYPERTENSIONAHA.116.07620

    Suppression of vascular EGFR activation in VSMC ADAM17 deficient mice. VSMC ADAM17 deficient mice and control mice were infused with AngII or saline as in . Tissues were immunostained with the antibodies indicated. Representative images are presented.
    Figure Legend Snippet: Suppression of vascular EGFR activation in VSMC ADAM17 deficient mice. VSMC ADAM17 deficient mice and control mice were infused with AngII or saline as in . Tissues were immunostained with the antibodies indicated. Representative images are presented.

    Techniques Used: Activation Assay, Mouse Assay

    9) Product Images from "N-Glycosylation of ss4 Integrin Controls the Adhesion and Motility of Keratinocytes"

    Article Title: N-Glycosylation of ss4 Integrin Controls the Adhesion and Motility of Keratinocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0027084

    N -glycosylation of ß4 integrin affects the N -glycosylation state of EGFR and galectin-3 mediated ß4 integrin-EGFR complex formation. (A) Cell surface expression levels of EGFR by FACS analysis against WT and ΔN keratinocytes. (B) Cell lysates from lac, WT and ΔN keratinocytes were immunoprecipitated using an anti-EGFR pAb. Immunoprecipitates were run on a 6% SDS-polyacrylamide gel and were probed with either the biotinylated E 4 -PHA lectin, L 4 -PHA lectin, DSA lectin and ConA lectin or an anti-EGFR Ab. (C) Covalent cross-linking was performed on WT and ΔN keratinocytes. After cross-linking, collected cell lysates were immunoprecipitated using an anti-EGFR pAb. Immunoprecipitates were run on a 5–20% SDS-polyacrylamide gel and were probed with the indicated Abs.
    Figure Legend Snippet: N -glycosylation of ß4 integrin affects the N -glycosylation state of EGFR and galectin-3 mediated ß4 integrin-EGFR complex formation. (A) Cell surface expression levels of EGFR by FACS analysis against WT and ΔN keratinocytes. (B) Cell lysates from lac, WT and ΔN keratinocytes were immunoprecipitated using an anti-EGFR pAb. Immunoprecipitates were run on a 6% SDS-polyacrylamide gel and were probed with either the biotinylated E 4 -PHA lectin, L 4 -PHA lectin, DSA lectin and ConA lectin or an anti-EGFR Ab. (C) Covalent cross-linking was performed on WT and ΔN keratinocytes. After cross-linking, collected cell lysates were immunoprecipitated using an anti-EGFR pAb. Immunoprecipitates were run on a 5–20% SDS-polyacrylamide gel and were probed with the indicated Abs.

    Techniques Used: Expressing, FACS, Immunoprecipitation

    Hypothetical model for the effect of N -glycans on ß4 integrin cellular function and signal transduction. Under normal conditions (WT ß4 integrin), ß4 integrin binds to EGFR and laminin-332 (Lm332) through cross-linking with galectin-3-mediated N -glycans, which induces a modest association among them, and thereby efficient integrin clustering, cellular signaling, and cell migration. However, in ΔNß4 integrin-expressing keratinocytes, the lack of galectin-3-meditaed ß4 integrin cross-linking and alternation in N -glycosylation of EGFR, resulted in a strong protein-protein association between ß4 integrin and EGFR, and a weak association between ß4 integrin and laminin-332, which inhibited efficient integrin clustering, thereby suppressing cellular signaling and cell migration. The N -glycan of ß4 integrin is also important for the lipid raft localization where SFKs-mediated phosphorylation takes place.
    Figure Legend Snippet: Hypothetical model for the effect of N -glycans on ß4 integrin cellular function and signal transduction. Under normal conditions (WT ß4 integrin), ß4 integrin binds to EGFR and laminin-332 (Lm332) through cross-linking with galectin-3-mediated N -glycans, which induces a modest association among them, and thereby efficient integrin clustering, cellular signaling, and cell migration. However, in ΔNß4 integrin-expressing keratinocytes, the lack of galectin-3-meditaed ß4 integrin cross-linking and alternation in N -glycosylation of EGFR, resulted in a strong protein-protein association between ß4 integrin and EGFR, and a weak association between ß4 integrin and laminin-332, which inhibited efficient integrin clustering, thereby suppressing cellular signaling and cell migration. The N -glycan of ß4 integrin is also important for the lipid raft localization where SFKs-mediated phosphorylation takes place.

    Techniques Used: Cell Function Assay, Transduction, Migration, Expressing

    10) Product Images from "G Protein-Coupled Receptor 30 Expression Is Up-Regulated by EGF and TGFα in Estrogen Receptor α-Positive Cancer Cells"

    Article Title: G Protein-Coupled Receptor 30 Expression Is Up-Regulated by EGF and TGFα in Estrogen Receptor α-Positive Cancer Cells

    Journal: Molecular Endocrinology

    doi: 10.1210/me.2009-0120

    EGF and TGFα induce EGFR phosphorylation and Erk1/2 activation in Ishikawa and MCF7-TR1 cells. Phosphorylation of EGFR Tyr 1045 and Tyr 1173 sites (A and B) and ERK1/2 (C) after a rapid treatment (5 min) with 100 ng/ml EGF or TGFα in Ishikawa and MCF7-TR1 cells. Total EGFR and ERK2 levels were used as a loading control. Data shown are representative of three independent experiments.
    Figure Legend Snippet: EGF and TGFα induce EGFR phosphorylation and Erk1/2 activation in Ishikawa and MCF7-TR1 cells. Phosphorylation of EGFR Tyr 1045 and Tyr 1173 sites (A and B) and ERK1/2 (C) after a rapid treatment (5 min) with 100 ng/ml EGF or TGFα in Ishikawa and MCF7-TR1 cells. Total EGFR and ERK2 levels were used as a loading control. Data shown are representative of three independent experiments.

    Techniques Used: Activation Assay

    11) Product Images from "Peritumoral Small EphrinA5 Isoform Level Predicts the Postoperative Survival in Hepatocellular Carcinoma"

    Article Title: Peritumoral Small EphrinA5 Isoform Level Predicts the Postoperative Survival in Hepatocellular Carcinoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0041749

    EphrinA5 isoforms suppressed EGFR expression by enhancing c-Cbl-mediated EGFR degradation. (A) Both ephrinA5L and ephrinA5S reduced EGFR protein expression level in Hep3B cells. Ectopic ephrinA5 reduced endogenous EGFR protein expression (left panel) but had no transcriptional modification of EGFR in RT-PCR (right panel). The differences were statistically significant between the treated group and untreated group. Experiments in each group were performed in triplicate. The level of significance was set at p
    Figure Legend Snippet: EphrinA5 isoforms suppressed EGFR expression by enhancing c-Cbl-mediated EGFR degradation. (A) Both ephrinA5L and ephrinA5S reduced EGFR protein expression level in Hep3B cells. Ectopic ephrinA5 reduced endogenous EGFR protein expression (left panel) but had no transcriptional modification of EGFR in RT-PCR (right panel). The differences were statistically significant between the treated group and untreated group. Experiments in each group were performed in triplicate. The level of significance was set at p

    Techniques Used: Expressing, Modification, Reverse Transcription Polymerase Chain Reaction

    12) Product Images from "Gi? proteins exhibit functional differences in the activation of ERK1/2, Akt and mTORC1 by growth factors in normal and breast cancer cells"

    Article Title: Gi? proteins exhibit functional differences in the activation of ERK1/2, Akt and mTORC1 by growth factors in normal and breast cancer cells

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/1478-811X-12-10

    G i α proteins associate with RTKs and their adaptor proteins in breast cancer cells , and are important for interactions of Gab1 with Shp2 or Grb2 in MEFs in response to EGF or bFGF. (A) MB231 cells were treated with bFGF (25 ng/ml) for 0, 2 or 5 min. Whole cell lysates were prepared. The associations of G i3 α with p-FGFR, Gab1 and FRS2 were determined by IB using anti-p-FGFR(Y766), anti-Gab1, anti-FRS2 and anti-G i3 α antibodies, respectively. (B-D) MB231 cells were treated with EGF (100 ng/ml) for 0, 2, 5, 10 or 20 min. The associations of G i3 α with p-EGFR, EGFR (B) , Gab1 and Shp2 (C D) were determined by IB using anti-p-EGFR (Y1173), anti-EGFR, anti-Gab1, anti-Shp2 and anti-G i3 α antibodies, respectively. (E F) WT and Gi1/3 DKO MEFs were treated with EGF or bFGF for 0, 2 or 5 min. The association of Shp2 with Gab1 in response to EGF (E) , and the associations of Grb2 with Gab1 and FRS2 in response to bFGF (F) were determined by IB. Experiments were repeated two to four times. Similar results were obtained.
    Figure Legend Snippet: G i α proteins associate with RTKs and their adaptor proteins in breast cancer cells , and are important for interactions of Gab1 with Shp2 or Grb2 in MEFs in response to EGF or bFGF. (A) MB231 cells were treated with bFGF (25 ng/ml) for 0, 2 or 5 min. Whole cell lysates were prepared. The associations of G i3 α with p-FGFR, Gab1 and FRS2 were determined by IB using anti-p-FGFR(Y766), anti-Gab1, anti-FRS2 and anti-G i3 α antibodies, respectively. (B-D) MB231 cells were treated with EGF (100 ng/ml) for 0, 2, 5, 10 or 20 min. The associations of G i3 α with p-EGFR, EGFR (B) , Gab1 and Shp2 (C D) were determined by IB using anti-p-EGFR (Y1173), anti-EGFR, anti-Gab1, anti-Shp2 and anti-G i3 α antibodies, respectively. (E F) WT and Gi1/3 DKO MEFs were treated with EGF or bFGF for 0, 2 or 5 min. The association of Shp2 with Gab1 in response to EGF (E) , and the associations of Grb2 with Gab1 and FRS2 in response to bFGF (F) were determined by IB. Experiments were repeated two to four times. Similar results were obtained.

    Techniques Used:

    13) Product Images from "Synergistic effect of targeting dishevelled-3 and the epidermal growth factor receptor-tyrosine kinase inhibitor on mesothelioma cells in vitro"

    Article Title: Synergistic effect of targeting dishevelled-3 and the epidermal growth factor receptor-tyrosine kinase inhibitor on mesothelioma cells in vitro

    Journal: Oncology Letters

    doi: 10.3892/ol.2017.7382

    Downregulation of Dvl-3 expression in mesothelioma cells using Dvl-3 siRNA. Western blot analysis revealed that expression of Dvl-3 was downregulated in 211H, H2452 and H28 cells after 48 h of transfection with Dvl-3 siRNA, compared with cells transfected with control siRNA. 211H and H2452 cells exhibited expression of EGFR and β-catenin, whereas H28 exhibited no β-catenin expression due to homozygous deletion of the β-catenin gene. β-actin served as a loading control. Dvl-3, dishevelled-3; siRNA, small interfering RNA; EGFR, epidermal growth factor receptor.
    Figure Legend Snippet: Downregulation of Dvl-3 expression in mesothelioma cells using Dvl-3 siRNA. Western blot analysis revealed that expression of Dvl-3 was downregulated in 211H, H2452 and H28 cells after 48 h of transfection with Dvl-3 siRNA, compared with cells transfected with control siRNA. 211H and H2452 cells exhibited expression of EGFR and β-catenin, whereas H28 exhibited no β-catenin expression due to homozygous deletion of the β-catenin gene. β-actin served as a loading control. Dvl-3, dishevelled-3; siRNA, small interfering RNA; EGFR, epidermal growth factor receptor.

    Techniques Used: Expressing, Western Blot, Transfection, Small Interfering RNA

    14) Product Images from "SPARC Overexpression Inhibits Cell Proliferation in Neuroblastoma and Is Partly Mediated by Tumor Suppressor Protein PTEN and AKT"

    Article Title: SPARC Overexpression Inhibits Cell Proliferation in Neuroblastoma and Is Partly Mediated by Tumor Suppressor Protein PTEN and AKT

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036093

    Constitutively active AKT overexpression blocks the radiosensitization capability of SPARC in neuroblastoma cells. SK-N-AS, NB1691 and IMR-32 cells were transfected with mock (PBS control), empty vector (pEV) or with pSPARC alone or in combination with plasmid overexpressing constitutively active AKT (myrAKT; pAKT) for 24 hours, and SK-N-AS and NB1691cells were irradiated with 8 Gy and IMR-32 cells were irradiated with 4 Gy dose of radiation and incubated for further 16 hours. ( A ) Cell lysates were assessed for SPARC, pAKT, EGFR, pEGFR, PTEN, c-Myc and Chk1 by western blotting. Protein levels were quantified by densitometric analysis and shown in the corresponding bar graph. Columns, mean of triplicate experiments; bars, SE. Results are representative of three independent experiments. ( B ) At the indicated time points, the plates were incubated by adding MTT reagent for a further 6 hours. The developed purple color formazan crystals were solubilized using DMSO, and color intensity was measured by spectrophotometer at 570 nm. Data are plotted at an absorbance of 570 nm. Results are representative of three independent experiments. Points, mean of triplicate experiments. H = hours.
    Figure Legend Snippet: Constitutively active AKT overexpression blocks the radiosensitization capability of SPARC in neuroblastoma cells. SK-N-AS, NB1691 and IMR-32 cells were transfected with mock (PBS control), empty vector (pEV) or with pSPARC alone or in combination with plasmid overexpressing constitutively active AKT (myrAKT; pAKT) for 24 hours, and SK-N-AS and NB1691cells were irradiated with 8 Gy and IMR-32 cells were irradiated with 4 Gy dose of radiation and incubated for further 16 hours. ( A ) Cell lysates were assessed for SPARC, pAKT, EGFR, pEGFR, PTEN, c-Myc and Chk1 by western blotting. Protein levels were quantified by densitometric analysis and shown in the corresponding bar graph. Columns, mean of triplicate experiments; bars, SE. Results are representative of three independent experiments. ( B ) At the indicated time points, the plates were incubated by adding MTT reagent for a further 6 hours. The developed purple color formazan crystals were solubilized using DMSO, and color intensity was measured by spectrophotometer at 570 nm. Data are plotted at an absorbance of 570 nm. Results are representative of three independent experiments. Points, mean of triplicate experiments. H = hours.

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Irradiation, Incubation, Western Blot, MTT Assay, Spectrophotometry

    SPARC overexpression inhibits AKT phosphorylation and induces PTEN. SK-N-AS, NB1691 and IMR-32 cells were transfected with mock (PBS control), empty vector (pEV) or with pSPARC for 24 hours, and SK-N-AS and NB1691cells were irradiated (IR) with 8 Gy and IMR-32 cells were irradiated with 4 Gy dose of radiation for further 16 hours. ( A ) Cell lysates were assessed for EGFR, AKT and their phosphorylations, PI3K, mTOR, PTEN and c-Myc by western blotting. ( B ) Protein levels were quantified by densitometric analysis for pAKT and PTEN is shown in the corresponding bar graph. Columns, mean of triplicate experiments; bars, SD. Results are representative of three independent experiments. * p
    Figure Legend Snippet: SPARC overexpression inhibits AKT phosphorylation and induces PTEN. SK-N-AS, NB1691 and IMR-32 cells were transfected with mock (PBS control), empty vector (pEV) or with pSPARC for 24 hours, and SK-N-AS and NB1691cells were irradiated (IR) with 8 Gy and IMR-32 cells were irradiated with 4 Gy dose of radiation for further 16 hours. ( A ) Cell lysates were assessed for EGFR, AKT and their phosphorylations, PI3K, mTOR, PTEN and c-Myc by western blotting. ( B ) Protein levels were quantified by densitometric analysis for pAKT and PTEN is shown in the corresponding bar graph. Columns, mean of triplicate experiments; bars, SD. Results are representative of three independent experiments. * p

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Irradiation, Western Blot

    15) Product Images from "TAK1-Mediated Serine/Threonine Phosphorylation of Epidermal Growth Factor Receptor via p38/Extracellular Signal-Regulated Kinase: NF-κB-Independent Survival Pathways in Tumor Necrosis Factor Alpha Signaling "

    Article Title: TAK1-Mediated Serine/Threonine Phosphorylation of Epidermal Growth Factor Receptor via p38/Extracellular Signal-Regulated Kinase: NF-κB-Independent Survival Pathways in Tumor Necrosis Factor Alpha Signaling

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00375-09

    EGFR is essential for protection from TNF-α-induced proapoptotic signals. (A) HeLa-shLuc or HeLa-shTAK1 cells were treated with TNF-α for 4 h. Whole-cell lysates were immunoblotted with phospho-JNK, JNK, PARP, TAK1, and tubulin antibodies. Arrows show cleaved forms of PARP. (B) HeLa cells were transfected with Luc siRNA or two different siRNAs against EGFR (#1 and #2). At 72 h posttransfection, cells were stimulated with TNF-α for 4 h. Whole-cell lysates were immunoblotted with caspase-3, PARP, EGFR, and actin antibodies. Arrows show cleaved forms of caspase-3 and PARP. (C and D) HeLa cells were pretreated with PD153035 (PD) (1 μM), 5Z-7-oxozeaenol (5Z) (0.3 μM), SB203580 (SB) (10 μM), or U0126 (U) (5 μM) for 30 min and then stimulated with TNF-α for another 4 h. Whole-cell lysates were immunoblotted with the indicated antibodies. DMSO, dimethyl sulfoxide. (E) HeLa cells were transfected with siRNAs against CHC, EGFR (#1), and Luc. At 72 h posttransfection, cells were stimulated with TNF-α for 4 h. Whole-cell lysates were immunoblotted with the indicated antibodies.
    Figure Legend Snippet: EGFR is essential for protection from TNF-α-induced proapoptotic signals. (A) HeLa-shLuc or HeLa-shTAK1 cells were treated with TNF-α for 4 h. Whole-cell lysates were immunoblotted with phospho-JNK, JNK, PARP, TAK1, and tubulin antibodies. Arrows show cleaved forms of PARP. (B) HeLa cells were transfected with Luc siRNA or two different siRNAs against EGFR (#1 and #2). At 72 h posttransfection, cells were stimulated with TNF-α for 4 h. Whole-cell lysates were immunoblotted with caspase-3, PARP, EGFR, and actin antibodies. Arrows show cleaved forms of caspase-3 and PARP. (C and D) HeLa cells were pretreated with PD153035 (PD) (1 μM), 5Z-7-oxozeaenol (5Z) (0.3 μM), SB203580 (SB) (10 μM), or U0126 (U) (5 μM) for 30 min and then stimulated with TNF-α for another 4 h. Whole-cell lysates were immunoblotted with the indicated antibodies. DMSO, dimethyl sulfoxide. (E) HeLa cells were transfected with siRNAs against CHC, EGFR (#1), and Luc. At 72 h posttransfection, cells were stimulated with TNF-α for 4 h. Whole-cell lysates were immunoblotted with the indicated antibodies.

    Techniques Used: Transfection

    Identification of phosphorylation sites on EGFR by overexpression experiments in HEK293 cells. (A) Structure of EGFR. EGFR is composed by extracellular, transmembrane (TM), JM, TK, and CR domains. Numbers represent the number of amino acids of EGFR that the N-terminal signal peptide (24 amino acids) processed. (B) HEK293 cells were transfected with expression vectors for EGFR and the TAK1/TAB1/TAB2 complex (TAK1-cpx). Twenty-four hours after transfection, cells were harvested and whole-cell lysates were immunoblotted with phospho-EGFR (Tyr-845, Tyr-992, Tyr-1045, Tyr-1068, and Tyr-1173), phosphotyrosine (PY20), EGFR, and TAK1, TAB1, and TAB2 antibodies. (C) Cells were transfected with expression vectors for the wild type (WT) or deletion mutants (Δ1113 and Δ1043) of EGFR and the TAK1 complex. Whole-cell lysates were immunoblotted with PY20 and EGFR antibodies. (D) Cells were transfected with expression vectors for the wild type or a kinase dead mutant (ΚΚ/ΑΑ) of EGFR and the TAK1 complex. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669 and Ser-1046/1047), PY20, EGFR, and TAK1 antibodies. (E and F) Cells were transfected with expression vectors for the wild type and substitution mutants (T669A or SS1046/1047AA) of full-length or deleted (Δ1075) EGFR and the TAK1 complex. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669 and Ser-1046/1047), EGFR, and TAK1 antibodies.
    Figure Legend Snippet: Identification of phosphorylation sites on EGFR by overexpression experiments in HEK293 cells. (A) Structure of EGFR. EGFR is composed by extracellular, transmembrane (TM), JM, TK, and CR domains. Numbers represent the number of amino acids of EGFR that the N-terminal signal peptide (24 amino acids) processed. (B) HEK293 cells were transfected with expression vectors for EGFR and the TAK1/TAB1/TAB2 complex (TAK1-cpx). Twenty-four hours after transfection, cells were harvested and whole-cell lysates were immunoblotted with phospho-EGFR (Tyr-845, Tyr-992, Tyr-1045, Tyr-1068, and Tyr-1173), phosphotyrosine (PY20), EGFR, and TAK1, TAB1, and TAB2 antibodies. (C) Cells were transfected with expression vectors for the wild type (WT) or deletion mutants (Δ1113 and Δ1043) of EGFR and the TAK1 complex. Whole-cell lysates were immunoblotted with PY20 and EGFR antibodies. (D) Cells were transfected with expression vectors for the wild type or a kinase dead mutant (ΚΚ/ΑΑ) of EGFR and the TAK1 complex. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669 and Ser-1046/1047), PY20, EGFR, and TAK1 antibodies. (E and F) Cells were transfected with expression vectors for the wild type and substitution mutants (T669A or SS1046/1047AA) of full-length or deleted (Δ1075) EGFR and the TAK1 complex. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669 and Ser-1046/1047), EGFR, and TAK1 antibodies.

    Techniques Used: Over Expression, Transfection, Expressing, Mutagenesis

    Schematic diagram of TNF-α-induced antiapoptotic signals. TNF-α binding to TNF-R1 rapidly induces the activation of TAK1. TAK1 induces two independent signaling pathways to EGFR through ERK and p38. TAK1 regulates two independent antiapoptotic pathways, NF-κB and p38-EGFR. These signals coordinately prevent death-inducing signaling complex (DISC)-mediated proapoptotic cleavage of caspase and PARP.
    Figure Legend Snippet: Schematic diagram of TNF-α-induced antiapoptotic signals. TNF-α binding to TNF-R1 rapidly induces the activation of TAK1. TAK1 induces two independent signaling pathways to EGFR through ERK and p38. TAK1 regulates two independent antiapoptotic pathways, NF-κB and p38-EGFR. These signals coordinately prevent death-inducing signaling complex (DISC)-mediated proapoptotic cleavage of caspase and PARP.

    Techniques Used: Binding Assay, Activation Assay

    Phosphorylation of endogenous EGFR at Thr-669 and Ser-1046/1047. (A) HeLa cells were treated with TNF-α or EGF for 10, 30, or 60 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669, Ser-1046/1047, Tyr-845, Tyr-974, Tyr-1045, Tyr-1068, and Tyr-1173), EGFR, phospho-MAPKs (p38, JNK, and ERK), and tubulin antibodies. (B) Cells were exposed to a high concentration of salt (Osmo) (300 mM NaCl), TNF-α, or EGF for the indicated time periods. Whole-cell lysates were immunoblotted with the indicated antibodies. (C) A549 cells were treated with TNF-α, IL-1β, or EGF for 10 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669 and Ser-1046/1047), EGFR, and actin antibodies. (D) HEK293 cells were transfected with expression vectors for the wild type (WT) and a kinase dead mutant (ΚΚ/ΑΑ) of EGFR. Twenty-four hours after transfection, cells were treated with TNF-α for 10 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669 and Ser-1046/1047) and EGFR antibodies. Cont, control.
    Figure Legend Snippet: Phosphorylation of endogenous EGFR at Thr-669 and Ser-1046/1047. (A) HeLa cells were treated with TNF-α or EGF for 10, 30, or 60 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669, Ser-1046/1047, Tyr-845, Tyr-974, Tyr-1045, Tyr-1068, and Tyr-1173), EGFR, phospho-MAPKs (p38, JNK, and ERK), and tubulin antibodies. (B) Cells were exposed to a high concentration of salt (Osmo) (300 mM NaCl), TNF-α, or EGF for the indicated time periods. Whole-cell lysates were immunoblotted with the indicated antibodies. (C) A549 cells were treated with TNF-α, IL-1β, or EGF for 10 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669 and Ser-1046/1047), EGFR, and actin antibodies. (D) HEK293 cells were transfected with expression vectors for the wild type (WT) and a kinase dead mutant (ΚΚ/ΑΑ) of EGFR. Twenty-four hours after transfection, cells were treated with TNF-α for 10 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669 and Ser-1046/1047) and EGFR antibodies. Cont, control.

    Techniques Used: Concentration Assay, Transfection, Expressing, Mutagenesis

    Signaling pathways leading to Ser/Thr phosphorylation of EGFR. (A) HeLa cells stably expressing shRNA against TAK1 (T) and luciferase (L) were stimulated with TNF-α or EGF for 10 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669, Ser-1046/1047, Tyr-845, 974, 1045, 1068 and 1173), EGFR, phospho-MAPKs (p38, JNK and ERK), and tubulin antibodies. Cont, control. (B and C) HeLa cells were pretreated with PD153035 (PD) (1 μM), 5Z-7-oxozeaenol (5Z) (0.3 μM), SB203580 (SB) (10 μM), U0126 (U) (5 μM), and SP600125 (SP) (10 μM) for 30 min and then stimulated with TNF-α (B) or EGF (C) for another 10 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669, Ser-1046/1047, and Tyr845), EGFR, and tubulin antibodies. (D) HeLa cells were transfected with siRNAs against ERK2, p38α, and Luc. At 72 h posttransfection, cells were stimulated with TNF-α for 10 min. Whole-cell lysates were immunoblotted with the indicated antibodies. (E) Cells were stimulated with TNF-α for the indicated time periods (min), and phosphorylation of EGFR, p38, and ERK was detected by immunoblotting.
    Figure Legend Snippet: Signaling pathways leading to Ser/Thr phosphorylation of EGFR. (A) HeLa cells stably expressing shRNA against TAK1 (T) and luciferase (L) were stimulated with TNF-α or EGF for 10 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669, Ser-1046/1047, Tyr-845, 974, 1045, 1068 and 1173), EGFR, phospho-MAPKs (p38, JNK and ERK), and tubulin antibodies. Cont, control. (B and C) HeLa cells were pretreated with PD153035 (PD) (1 μM), 5Z-7-oxozeaenol (5Z) (0.3 μM), SB203580 (SB) (10 μM), U0126 (U) (5 μM), and SP600125 (SP) (10 μM) for 30 min and then stimulated with TNF-α (B) or EGF (C) for another 10 min. Whole-cell lysates were immunoblotted with phospho-EGFR (Thr-669, Ser-1046/1047, and Tyr845), EGFR, and tubulin antibodies. (D) HeLa cells were transfected with siRNAs against ERK2, p38α, and Luc. At 72 h posttransfection, cells were stimulated with TNF-α for 10 min. Whole-cell lysates were immunoblotted with the indicated antibodies. (E) Cells were stimulated with TNF-α for the indicated time periods (min), and phosphorylation of EGFR, p38, and ERK was detected by immunoblotting.

    Techniques Used: Stable Transfection, Expressing, shRNA, Luciferase, Transfection

    NF-κB is not involved in EGFR phosphorylation. (A) HeLa-shLuc or HeLa-shTAK1 cells were treated with TNF-α for 5 min. Whole-cell lysates were immunoblotted with phospho-p65, p65, PARP, TAK1, and tubulin antibodies. (B to D) HeLa cells were transfected with siRNAs against p65, EGFR (#1), and Luc. At 72 h posttransfection, cells were stimulated with TNF-α for the indicated time periods (B and C) or 4 h (D). Whole-cell lysates were immunoblotted with the indicated antibodies.
    Figure Legend Snippet: NF-κB is not involved in EGFR phosphorylation. (A) HeLa-shLuc or HeLa-shTAK1 cells were treated with TNF-α for 5 min. Whole-cell lysates were immunoblotted with phospho-p65, p65, PARP, TAK1, and tubulin antibodies. (B to D) HeLa cells were transfected with siRNAs against p65, EGFR (#1), and Luc. At 72 h posttransfection, cells were stimulated with TNF-α for the indicated time periods (B and C) or 4 h (D). Whole-cell lysates were immunoblotted with the indicated antibodies.

    Techniques Used: Transfection

    16) Product Images from "Ectodomain cleavage of FLT1 regulates receptor activation and function and is not required for its downstream intracellular cleavage"

    Article Title: Ectodomain cleavage of FLT1 regulates receptor activation and function and is not required for its downstream intracellular cleavage

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2016.03.020

    Quantitation of cFlt1, surface expression and dimerization of Flt1 mutant proteins and cleavage of Flt1-KDR hybrid proteins. Panel A: Free VEGF measured by ELISA in conditioned media (CM) from HEK293 cells transiently transfected with pcDNA3, FLT1 or various FLT1 deletion constructs. The cleaved Flt1 ectodomain binds to secreted VEGF and the reduction in free VEGF in CM reflects the abundance of the cleaved ectodomain. Compared to wild type Flt1, the FLT1 deletion constructs Δ699-763, Δ754-763 and Δ759-763 have markedly diminished ability to reduce free VEGF. Panel B: Expression of Flt1 and various mutant Flt1 proteins were assessed in total lysates and surface fractions of transfected HEK293 cells. Lysates and affinity purified surface proteins were probed with Flag antibody. Endogenous tubulin and EGFR were used as loading controls. Panel C: COS7 cells transiently transfected with pcDNA3, FLT1 or Δ754-763 was subject to crosslinking with DSS or exposed to vehicle and then cell lysates probed with FLAG or EGFR as a loading control. A shift in Flt1 and Δ754-763 from the monomeric to the dimeric form is seen with DSS in the FLAG immunoblot and a corresponding shift in size is seen for the EGFR bands. Panel D: HEK293 cells transiently transfected with pcDNA3, KDR, or KDR-Flt1 hybrids and the cleaved HA tagged ectodomain probed by immunoblotting in conditioned media and tGFP-tagged KDR and KDR-Flt1 hybrid proteins probed in total cell lysates. Tubulin was used as a loading control.
    Figure Legend Snippet: Quantitation of cFlt1, surface expression and dimerization of Flt1 mutant proteins and cleavage of Flt1-KDR hybrid proteins. Panel A: Free VEGF measured by ELISA in conditioned media (CM) from HEK293 cells transiently transfected with pcDNA3, FLT1 or various FLT1 deletion constructs. The cleaved Flt1 ectodomain binds to secreted VEGF and the reduction in free VEGF in CM reflects the abundance of the cleaved ectodomain. Compared to wild type Flt1, the FLT1 deletion constructs Δ699-763, Δ754-763 and Δ759-763 have markedly diminished ability to reduce free VEGF. Panel B: Expression of Flt1 and various mutant Flt1 proteins were assessed in total lysates and surface fractions of transfected HEK293 cells. Lysates and affinity purified surface proteins were probed with Flag antibody. Endogenous tubulin and EGFR were used as loading controls. Panel C: COS7 cells transiently transfected with pcDNA3, FLT1 or Δ754-763 was subject to crosslinking with DSS or exposed to vehicle and then cell lysates probed with FLAG or EGFR as a loading control. A shift in Flt1 and Δ754-763 from the monomeric to the dimeric form is seen with DSS in the FLAG immunoblot and a corresponding shift in size is seen for the EGFR bands. Panel D: HEK293 cells transiently transfected with pcDNA3, KDR, or KDR-Flt1 hybrids and the cleaved HA tagged ectodomain probed by immunoblotting in conditioned media and tGFP-tagged KDR and KDR-Flt1 hybrid proteins probed in total cell lysates. Tubulin was used as a loading control.

    Techniques Used: Quantitation Assay, Expressing, Mutagenesis, Enzyme-linked Immunosorbent Assay, Transfection, Construct, Affinity Purification

    17) Product Images from "Distinct role of PLC?3 in VEGF-mediated directional migration and vascular sprouting"

    Article Title: Distinct role of PLC?3 in VEGF-mediated directional migration and vascular sprouting

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.041913

    Specificity of VEGFR induces serine phosphorylation of PLCβ3. (A) Serum-starved HUVECs were pretreated with or without kinase inhibitor (100 nM) and then stimulated with or without 10 mg/ml VEGF for 5 minutes. Immunoblotting was performed with antibodies as shown. (B) HUVECs were infected with or without retrovirus expressing EGDR or EGLT for 48 hours. Tyrosine phosphorylation of EGDR or EGLT following EGF treatment was confirmed by immunoprecipitation with an N-terminal EGFR antibody followed by immunoblotting with antibody against Tyr- P . Expression of EGDR and EGLT was confirmed by immunoprecipitation with an N-terminal EGFR antibody followed by immunoblotting with antibodies against KDR or Flt-1. (C) HUVECs were infected with retrovirus expressing EGDR or EGLT for 48 hours, followed by treatment with EGF (10 ng/ml) and immunoblotting as shown.
    Figure Legend Snippet: Specificity of VEGFR induces serine phosphorylation of PLCβ3. (A) Serum-starved HUVECs were pretreated with or without kinase inhibitor (100 nM) and then stimulated with or without 10 mg/ml VEGF for 5 minutes. Immunoblotting was performed with antibodies as shown. (B) HUVECs were infected with or without retrovirus expressing EGDR or EGLT for 48 hours. Tyrosine phosphorylation of EGDR or EGLT following EGF treatment was confirmed by immunoprecipitation with an N-terminal EGFR antibody followed by immunoblotting with antibody against Tyr- P . Expression of EGDR and EGLT was confirmed by immunoprecipitation with an N-terminal EGFR antibody followed by immunoblotting with antibodies against KDR or Flt-1. (C) HUVECs were infected with retrovirus expressing EGDR or EGLT for 48 hours, followed by treatment with EGF (10 ng/ml) and immunoblotting as shown.

    Techniques Used: Infection, Expressing, Immunoprecipitation

    18) Product Images from "Suppression of Androgen Receptor Enhances the Self-Renewal of Mesenchymal Stem Cells Through Elevated Expression of EGFR"

    Article Title: Suppression of Androgen Receptor Enhances the Self-Renewal of Mesenchymal Stem Cells Through Elevated Expression of EGFR

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamcr.2013.01.007

    The activation of Akt and Erk signaling in AR knocked out BM-MSCs is through EGF/EGFR
    Figure Legend Snippet: The activation of Akt and Erk signaling in AR knocked out BM-MSCs is through EGF/EGFR

    Techniques Used: Activation Assay

    19) Product Images from "Nuclear EGFR Contributes to Acquired Resistance to Cetuximab"

    Article Title: Nuclear EGFR Contributes to Acquired Resistance to Cetuximab

    Journal: Oncogene

    doi: 10.1038/onc.2009.234

    Overexpression of a NLS tagged EGFR in cetuximab-resistant cells confers resistance to cetuximab in vivo Male athymic nude mice were injected subcutaneously with 1×10 6 cetuximab-sensitive parental cells (HP) or CMV-EGFR-NLS/Myc clone cells (Vector only, Clone 4, Clone 5 and Clone 10) into the dorsal flank. Once tumors reached a volume 120-180mm 3 mice were treated with 0.1 mg IgG or cetuximab twice weekly. Tumor diameters were measured serially with calipers and tumor volumes were calculated. Points, mean tumor volume of eight mice per group; bars, SD. T -test was used to compare tumor volumes between cetuximab treated and control IgG mice. *, P
    Figure Legend Snippet: Overexpression of a NLS tagged EGFR in cetuximab-resistant cells confers resistance to cetuximab in vivo Male athymic nude mice were injected subcutaneously with 1×10 6 cetuximab-sensitive parental cells (HP) or CMV-EGFR-NLS/Myc clone cells (Vector only, Clone 4, Clone 5 and Clone 10) into the dorsal flank. Once tumors reached a volume 120-180mm 3 mice were treated with 0.1 mg IgG or cetuximab twice weekly. Tumor diameters were measured serially with calipers and tumor volumes were calculated. Points, mean tumor volume of eight mice per group; bars, SD. T -test was used to compare tumor volumes between cetuximab treated and control IgG mice. *, P

    Techniques Used: Over Expression, In Vivo, Mouse Assay, Injection, Plasmid Preparation

    Potential mechanism for resistance to cetuximab A ) Cetuximab-sensitive cells depend on classical EGFR membrane signaling. B ) Tumor cells that acquire resistance to cetuximab gain nEGFR as a second compartment of proliferation. C ) Cetuximab can abrogate signals from plasma membrane EGFR but not nEGFR; nEGFR continues to send proliferative signals by modulation of Cyclin D1, B-myb, Aurora kinase K and regulation of PCNA. D ) The SFK inhibitor dasatinib inhibits nuclear translocation of the EGFR from the plasma membrane leading to increased EGFR on the plasma membrane and restoring sensitivity to cetuximab.
    Figure Legend Snippet: Potential mechanism for resistance to cetuximab A ) Cetuximab-sensitive cells depend on classical EGFR membrane signaling. B ) Tumor cells that acquire resistance to cetuximab gain nEGFR as a second compartment of proliferation. C ) Cetuximab can abrogate signals from plasma membrane EGFR but not nEGFR; nEGFR continues to send proliferative signals by modulation of Cyclin D1, B-myb, Aurora kinase K and regulation of PCNA. D ) The SFK inhibitor dasatinib inhibits nuclear translocation of the EGFR from the plasma membrane leading to increased EGFR on the plasma membrane and restoring sensitivity to cetuximab.

    Techniques Used: Translocation Assay

    Src family kinases mediate ligand-induced EGFR translocation to the nucleus A ) Dasatinib inhibits HER family ligands signaling in parental cells (HP). HP cells were untreated, treated for 24 hours with 50 nM of dasatinib alone, or followed by 200 ng/ml of indicated ligand for 1 hour prior to harvesting. Nuclear protein was collected and fractionated by SDS-PAGE followed by immunoblotting for EGFR. histone H3 was used as loading control. B ) Dasatinib inhibits nuclear expression of EGFR in cetuximab-resistant cell lines. Parental cells (HP) and cetuximab-resistant cell lines (HC1, HC4, HC8) were treated with 50 nM of dasatinib for 24 hours. After cells were harvested, cytoplasmic and nuclear protein was fractionated by SDS-PAGE followed by immunoblotting for EGFR. α-tubulin and histone H3 were used as loading controls and purity controls of each cellular fraction. Expression of nEGFR after dasatinib treatment in cetuximab-resistant clones was quantitated using ImageJ software and normalized against the amounts of untreated cells. C ) Dasatinib treatment lead to increased membrane-bound EGFR in cetuximab-resistant cells by flow cytometry analysis. Parental cells (HP) and cetuximab-resistant cells (HC1, HC4 and HC8) were treated with DMSO or 50 nM of dasatinib for 24 hours and membrane expression is represented relative to untreated controls. Mean surface expression of EGFR is represented +/- SEM (n=3). Flow cytometric plots of representative experiments are presented. Shaded histograms represent dasatinib treatment. Controls (dotted line) represent cells labeled with FITC-conjugated normal mouse IgG *, P
    Figure Legend Snippet: Src family kinases mediate ligand-induced EGFR translocation to the nucleus A ) Dasatinib inhibits HER family ligands signaling in parental cells (HP). HP cells were untreated, treated for 24 hours with 50 nM of dasatinib alone, or followed by 200 ng/ml of indicated ligand for 1 hour prior to harvesting. Nuclear protein was collected and fractionated by SDS-PAGE followed by immunoblotting for EGFR. histone H3 was used as loading control. B ) Dasatinib inhibits nuclear expression of EGFR in cetuximab-resistant cell lines. Parental cells (HP) and cetuximab-resistant cell lines (HC1, HC4, HC8) were treated with 50 nM of dasatinib for 24 hours. After cells were harvested, cytoplasmic and nuclear protein was fractionated by SDS-PAGE followed by immunoblotting for EGFR. α-tubulin and histone H3 were used as loading controls and purity controls of each cellular fraction. Expression of nEGFR after dasatinib treatment in cetuximab-resistant clones was quantitated using ImageJ software and normalized against the amounts of untreated cells. C ) Dasatinib treatment lead to increased membrane-bound EGFR in cetuximab-resistant cells by flow cytometry analysis. Parental cells (HP) and cetuximab-resistant cells (HC1, HC4 and HC8) were treated with DMSO or 50 nM of dasatinib for 24 hours and membrane expression is represented relative to untreated controls. Mean surface expression of EGFR is represented +/- SEM (n=3). Flow cytometric plots of representative experiments are presented. Shaded histograms represent dasatinib treatment. Controls (dotted line) represent cells labeled with FITC-conjugated normal mouse IgG *, P

    Techniques Used: Translocation Assay, SDS Page, Expressing, Clone Assay, Software, Flow Cytometry, Cytometry, Labeling

    EGFR tagged with nuclear localization sequence confers resistance to cetuximab in vitro A ) A schematic representation of the CMV-EGFR-NLS/Myc construct is shown. EGFR-NLS/Myc was driven by the CMV promoter. The cetuximab-sensitive NSCLC line NCI-H226 was infected with indicated constructs. Represented is three individual clones and vector control (V 0 ; vector only, C 4 , C 5 and C 10 ). Cytoplasmic and nuclear protein from each clone was collected and immunoprecipitated with an anti-myc antibody, fractionated on SDS-PAGE and immunoblotted with the indicated antibodies. α-tubulin and histone H3 were used as loading and purity control for cytosolic and nuclear fractions, respectively. Immunofluorescence of nEGFR staining in CMV-EGFR-NLS/Myc clones. EGFR (green), DNA (blue), stained by PI. V 0 ; vector clone, C 4 , C 5 and C 10 ; CMV-EGFR-NLS/Myc clones. 400× magnification. cEGFR; cytoplasmic EGFR, nEGFR; nuclear EGFR. B ) CMV-EGFR-NLS/Myc expressed in NCI-H226 leads to increased cyclin D1 and B-myb expression. Nuclear protein from EGFR-NLS/myc clones was collected and fractionated by SDS-PAGE followed by immunoblotting for the indicated proteins. histone H3 was used as a loading control. Expression of cyclin D1 and B-myb in CMV-EGFR-NLS/Myc clones (C 4 , C 5 and C 10 ) were quantitated using ImageJ software and normalized against the amounts of those proteins in vector control (V 0 ). V 0 ; vector clone, C 4 , C 5 and C 10 ; CMV-EGFR-NLS/Myc clones. C ) Growth response to cetuximab of three individual clones and vector control (V 0 ; vector only, C 4 , C 5 and C 10 ). CMV-EGFR-NLS/Myc-tag clones (C 4 , C 5 and C 10 ) were treated with 100 nM of cetuximab and growth was measured using the growth proliferation assay and plotted as growth relative to untreated control. Data points are represented as mean +/- SEM. (n=3). *, P
    Figure Legend Snippet: EGFR tagged with nuclear localization sequence confers resistance to cetuximab in vitro A ) A schematic representation of the CMV-EGFR-NLS/Myc construct is shown. EGFR-NLS/Myc was driven by the CMV promoter. The cetuximab-sensitive NSCLC line NCI-H226 was infected with indicated constructs. Represented is three individual clones and vector control (V 0 ; vector only, C 4 , C 5 and C 10 ). Cytoplasmic and nuclear protein from each clone was collected and immunoprecipitated with an anti-myc antibody, fractionated on SDS-PAGE and immunoblotted with the indicated antibodies. α-tubulin and histone H3 were used as loading and purity control for cytosolic and nuclear fractions, respectively. Immunofluorescence of nEGFR staining in CMV-EGFR-NLS/Myc clones. EGFR (green), DNA (blue), stained by PI. V 0 ; vector clone, C 4 , C 5 and C 10 ; CMV-EGFR-NLS/Myc clones. 400× magnification. cEGFR; cytoplasmic EGFR, nEGFR; nuclear EGFR. B ) CMV-EGFR-NLS/Myc expressed in NCI-H226 leads to increased cyclin D1 and B-myb expression. Nuclear protein from EGFR-NLS/myc clones was collected and fractionated by SDS-PAGE followed by immunoblotting for the indicated proteins. histone H3 was used as a loading control. Expression of cyclin D1 and B-myb in CMV-EGFR-NLS/Myc clones (C 4 , C 5 and C 10 ) were quantitated using ImageJ software and normalized against the amounts of those proteins in vector control (V 0 ). V 0 ; vector clone, C 4 , C 5 and C 10 ; CMV-EGFR-NLS/Myc clones. C ) Growth response to cetuximab of three individual clones and vector control (V 0 ; vector only, C 4 , C 5 and C 10 ). CMV-EGFR-NLS/Myc-tag clones (C 4 , C 5 and C 10 ) were treated with 100 nM of cetuximab and growth was measured using the growth proliferation assay and plotted as growth relative to untreated control. Data points are represented as mean +/- SEM. (n=3). *, P

    Techniques Used: Sequencing, In Vitro, Construct, Infection, Clone Assay, Plasmid Preparation, Immunoprecipitation, SDS Page, Immunofluorescence, Staining, Expressing, Software, Proliferation Assay

    20) Product Images from "Repertaxin, an inhibitor of the chemokine receptors CXCR1 and CXCR2, inhibits malignant behavior of human gastric cancer MKN45 cells in vitro and in vivo and enhances efficacy of 5-fluorouracil"

    Article Title: Repertaxin, an inhibitor of the chemokine receptors CXCR1 and CXCR2, inhibits malignant behavior of human gastric cancer MKN45 cells in vitro and in vivo and enhances efficacy of 5-fluorouracil

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2016.3371

    Effects of repertaxin and repertaxin combined with 5-FU on cell proliferation, cell cycle, cell apoptosis, cell migration and invasion-related signaling molecules in the MKN45 cells. MKN45 cells were treated with repertaxin (25 μg/ml) alone, 5-FU (10 μg/ml) alone, or combined repertaxin and 5-FU for 48 h. (A) angiogenesis (VEGF), (B) apoptosis (Bcl-2 and Bax), (C) proliferation and growth (cyclin D1, EGFR and Ki-67), (D) invasion and metastasis (MMP-9, MMP-2, TIMP-2 and E-cadherin). mRNA expression was determined by real-time RT-PCR. GAPDH was used as an internal control. (E) Cell lysates (50 μg) were fractionated by SDS-PAGE and subject to western blot analysis; GAPDH was used as a loading control. Data are shown as mean ± SD. * P
    Figure Legend Snippet: Effects of repertaxin and repertaxin combined with 5-FU on cell proliferation, cell cycle, cell apoptosis, cell migration and invasion-related signaling molecules in the MKN45 cells. MKN45 cells were treated with repertaxin (25 μg/ml) alone, 5-FU (10 μg/ml) alone, or combined repertaxin and 5-FU for 48 h. (A) angiogenesis (VEGF), (B) apoptosis (Bcl-2 and Bax), (C) proliferation and growth (cyclin D1, EGFR and Ki-67), (D) invasion and metastasis (MMP-9, MMP-2, TIMP-2 and E-cadherin). mRNA expression was determined by real-time RT-PCR. GAPDH was used as an internal control. (E) Cell lysates (50 μg) were fractionated by SDS-PAGE and subject to western blot analysis; GAPDH was used as a loading control. Data are shown as mean ± SD. * P

    Techniques Used: Migration, Expressing, Quantitative RT-PCR, SDS Page, Western Blot

    21) Product Images from "Betacellulin (BTC) Biases the EGFR To Dimerize with ErbB3"

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

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.118.113399

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

    Techniques Used: Multiple Displacement Amplification

    22) Product Images from "The Synergistic Effect of Combination Progesterone and Temozolomide on Human Glioblastoma Cells"

    Article Title: The Synergistic Effect of Combination Progesterone and Temozolomide on Human Glioblastoma Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0131441

    Effect of PROG and TMZ on the proliferation and the expression of MGMT in U87MG and U118MG cells. Tumor cells (U87MG and U118MG) were seeded (0.5 x 10 6 ) in 60-mm petri dishes and kept under starvation overnight prior to drug exposure. Cells were repeatedly exposed to different concentrations of PROG and/or TMZ for 3 days. Protein samples (50 μg) were separated under reducing and denaturing conditions by 4–20% acrylamide Criterion gel and analyzed for EGFR, pAkt, total Akt and mTOR expression. The density of each P-Akt band was corrected for variance in loading, using the density of the corresponding total Akt. β-actin was used as a loading control for densitometry. (A) Representative Western blot and densitometric analysis of the expression of proliferation marker PCNA. (B) Expression of MGMT in U87MG and U118MG cells, and (C) Inhibitory effect of PROG on MGMT in U118MG cells. Data are expressed as means ± SD from two separate replication experiments (n = 3 samples each). Statistically significant difference: * P
    Figure Legend Snippet: Effect of PROG and TMZ on the proliferation and the expression of MGMT in U87MG and U118MG cells. Tumor cells (U87MG and U118MG) were seeded (0.5 x 10 6 ) in 60-mm petri dishes and kept under starvation overnight prior to drug exposure. Cells were repeatedly exposed to different concentrations of PROG and/or TMZ for 3 days. Protein samples (50 μg) were separated under reducing and denaturing conditions by 4–20% acrylamide Criterion gel and analyzed for EGFR, pAkt, total Akt and mTOR expression. The density of each P-Akt band was corrected for variance in loading, using the density of the corresponding total Akt. β-actin was used as a loading control for densitometry. (A) Representative Western blot and densitometric analysis of the expression of proliferation marker PCNA. (B) Expression of MGMT in U87MG and U118MG cells, and (C) Inhibitory effect of PROG on MGMT in U118MG cells. Data are expressed as means ± SD from two separate replication experiments (n = 3 samples each). Statistically significant difference: * P

    Techniques Used: Expressing, Western Blot, Marker

    Effect of PROG and TMZ on the PI3k/Akt/mTOR signaling pathway in U87MG and U118MG cells. Tumor cells (U87MG and U118MG) were seeded (0.5 x 10 6 ) in 60-mm petri dishes and kept under starvation overnight prior to drug exposure. Cells were repeatedly exposed to different concentrations of PROG and/or TMZ for 3 days. Protein samples (50 μg) were separated under reducing and denaturing conditions by 4–20% acrylamide Criterion gel and analyzed for EGFR, pAkt, total Akt and mTOR expression. The density of each P-Akt band was normalized with the density of corresponding total Akt band. β-actin was used as a loading control for densitometry. Representative Western blot and densitometric analysis of the expression of EGFR, phospho-Akt (Ser473) and mTOR in (A) U87MG and (B) U118MG cell lines. Data are expressed as means ± SD from two separate replication experiments (n = 3 samples each). Statistically significant difference: * P
    Figure Legend Snippet: Effect of PROG and TMZ on the PI3k/Akt/mTOR signaling pathway in U87MG and U118MG cells. Tumor cells (U87MG and U118MG) were seeded (0.5 x 10 6 ) in 60-mm petri dishes and kept under starvation overnight prior to drug exposure. Cells were repeatedly exposed to different concentrations of PROG and/or TMZ for 3 days. Protein samples (50 μg) were separated under reducing and denaturing conditions by 4–20% acrylamide Criterion gel and analyzed for EGFR, pAkt, total Akt and mTOR expression. The density of each P-Akt band was normalized with the density of corresponding total Akt band. β-actin was used as a loading control for densitometry. Representative Western blot and densitometric analysis of the expression of EGFR, phospho-Akt (Ser473) and mTOR in (A) U87MG and (B) U118MG cell lines. Data are expressed as means ± SD from two separate replication experiments (n = 3 samples each). Statistically significant difference: * P

    Techniques Used: Expressing, Western Blot

    Related Articles

    Transfection:

    Article Title: Novel EGFR inhibitors attenuate cardiac hypertrophy induced by angiotensin II
    Article Snippet: The cells were cultured in DMEM/F12 supplemented with 10% foetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin at 37°C in a humidified 5% CO2 atmosphere. .. Transient transfection of EGFR shRNA The small hairpin RNA (shRNA) specifically targeting the nucleotides of EGFR and its control shRNA contained in the plasmid were obtained from Santa Cruz Biotech. .. H9c2 cells were transiently transfected with shRNAs (1 μg/ml) using PolyJet transfection reagent (SignaGen Laboratories, Ljamsville, MD, USA) at a ratio of 3 μl PolyJet to 1 μg plasmid in DMEM/F12 containing 10% fetal bovine serum (FBS) for 48 hrs according to the manufacturer's instructions.

    shRNA:

    Article Title: Novel EGFR inhibitors attenuate cardiac hypertrophy induced by angiotensin II
    Article Snippet: The cells were cultured in DMEM/F12 supplemented with 10% foetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin at 37°C in a humidified 5% CO2 atmosphere. .. Transient transfection of EGFR shRNA The small hairpin RNA (shRNA) specifically targeting the nucleotides of EGFR and its control shRNA contained in the plasmid were obtained from Santa Cruz Biotech. .. H9c2 cells were transiently transfected with shRNAs (1 μg/ml) using PolyJet transfection reagent (SignaGen Laboratories, Ljamsville, MD, USA) at a ratio of 3 μl PolyJet to 1 μg plasmid in DMEM/F12 containing 10% fetal bovine serum (FBS) for 48 hrs according to the manufacturer's instructions.

    Plasmid Preparation:

    Article Title: Novel EGFR inhibitors attenuate cardiac hypertrophy induced by angiotensin II
    Article Snippet: The cells were cultured in DMEM/F12 supplemented with 10% foetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin at 37°C in a humidified 5% CO2 atmosphere. .. Transient transfection of EGFR shRNA The small hairpin RNA (shRNA) specifically targeting the nucleotides of EGFR and its control shRNA contained in the plasmid were obtained from Santa Cruz Biotech. .. H9c2 cells were transiently transfected with shRNAs (1 μg/ml) using PolyJet transfection reagent (SignaGen Laboratories, Ljamsville, MD, USA) at a ratio of 3 μl PolyJet to 1 μg plasmid in DMEM/F12 containing 10% fetal bovine serum (FBS) for 48 hrs according to the manufacturer's instructions.

    Western Blot:

    Article Title: Acquired resistance to 5-fluorouracil via HSP90/Src-mediated increase in thymidylate synthase expression in colon cancer
    Article Snippet: Migration and invasion assay Migration and invasion assays were performed as described previously [ ]. .. Western blotting Total protein isolation and Western blotting analyses were done as described previously [ ] with specific antibodies against the following antigens: pIGF-1R/IR (Y1131), IR, pEGFR (Y845), Src, pSrc (Y416), pp70S6K, p70S6K, pmTOR, mTOR, pAkt, Akt, pERK1/2 (p42/44), MEK1/2, ERK, tubulin (Cell Signaling Technology, Danvers, MA, USA); PARP, pFAK (Y397), and FAK (BD Biosciences, San Jose, CA, USA); Actin, IGF-1R (C-20), EGFR (1005), and Myc (9E10) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); TYMS (Invitrogen, Carlsbad, CA, USA). .. Reverse transcription polymerase chain reaction (RT-PCR) Total RNA extraction and reverse transcription was done as described previously [ ].

    Article Title: Taurocholate Induces Cyclooxygenase-2 Expression via the Sphingosine 1-phosphate Receptor 2 in a Human Cholangiocarcinoma Cell Line *
    Article Snippet: .. Primary antibodies against COX-2, p-ERK1/2, ERK1, ERK2, p-Akt, Akt, p-IKKα/β, IKKα/β, p-NF-κB p65, NF-κB p65, p-EGFR, EGFR, β-actin, and lamin B, as well as HRP-conjugated secondary antibodies for Western blot were from Santa Cruz Biotechnology (Santa Cruz, CA). .. Alexa Fluor® 488-conjugated secondary antibodies for immunofluorescence, High-capacity cDNA Reverse Transcription Kit, Lipofectamine 2000, cell cultures medium, and supplement components were all from Life Technologies (Grand Island, NY).

    Isolation:

    Article Title: Acquired resistance to 5-fluorouracil via HSP90/Src-mediated increase in thymidylate synthase expression in colon cancer
    Article Snippet: Migration and invasion assay Migration and invasion assays were performed as described previously [ ]. .. Western blotting Total protein isolation and Western blotting analyses were done as described previously [ ] with specific antibodies against the following antigens: pIGF-1R/IR (Y1131), IR, pEGFR (Y845), Src, pSrc (Y416), pp70S6K, p70S6K, pmTOR, mTOR, pAkt, Akt, pERK1/2 (p42/44), MEK1/2, ERK, tubulin (Cell Signaling Technology, Danvers, MA, USA); PARP, pFAK (Y397), and FAK (BD Biosciences, San Jose, CA, USA); Actin, IGF-1R (C-20), EGFR (1005), and Myc (9E10) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); TYMS (Invitrogen, Carlsbad, CA, USA). .. Reverse transcription polymerase chain reaction (RT-PCR) Total RNA extraction and reverse transcription was done as described previously [ ].

    Incubation:

    Article Title: A phosphorylation defective retinoic acid receptor mutant mimics the effects of retinoic acid on EGFR mediated AP-1 expression and cancer cell proliferation
    Article Snippet: Separated proteins were electroblotted to PVDF membranes according to manufacturer's recommendations (Roche Molecular Biochemicals). .. Blots were incubated with antibodies to human cyclin H, cdk7, cyclin A, p21WAF1/Cip1 , p27Kip1 , EGFR, activated and total ERK1, c-fos, Fra-1, c-jun, JunB, or β-actin (Santa Cruz Biotechnology) for 16 hours at 4°C. .. After washing in Tris buffered saline containing 0.1% Tween 20 (TBST, pH 7.4), blots were incubated for 30 minutes at room temperature with anti-IgG secondary antibody conjugated to horseradish peroxidase.

    Article Title: CD73/NT5E is a target of miR-30a-5p and plays an important role in the pathogenesis of non-small cell lung cancer
    Article Snippet: Immunohistochemical assay Immunohistochemical (IHC) analyses of tissues were conducted as described in our previous study [ ]. .. In brief, sections were incubated with EGFR (A-10) and CD73 (IE9) specific monoclonal primary antibodies (diluted 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C, then incubated with biotinylated secondary antibodies. .. The reactions were developed using the DAB Kit (BD Bioscience, San Jose, CA, USA) and the sections were counterstained with hematoxylin.

    Article Title: Paricalcitol Inhibits Aldosterone-Induced Proinflammatory Factors by Modulating Epidermal Growth Factor Receptor Pathway in Cultured Tubular Epithelial Cells
    Article Snippet: .. Samples were transferred to nitrocellulose membranes (Bio-Rad), blocked in 50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 0.05% Tween-20, and 5% milk, and incubated overnight at 4°C with the following antibodies [dilution]: anti-phosphorylated-EGFR on Tyrosine (Y) 1068 (p-EGFR1068 ) [1 : 250] (Calbiochem), ADAM17 [1 : 1000] (Abcam), EGFR [1 : 250], p-ERK1/2 [1 : 200] (Santa Cruz Biotechnology), and p-STAT1 [1 : 500] (Invitrogen). .. Subsequently, membranes were incubated with a peroxidase-conjugated secondary antibody and developed using the ECL chemiluminescence kit (Amersham Pharmacia Biotech).

    other:

    Article Title: Nuclear Gene 33/Mig6 regulates the DNA damage response through an ATM serine/threonine kinase–dependent mechanism
    Article Snippet: Antibodies to GAPDH, EGFR, Mig6, and β-actin were obtained from Santa Cruz Biotechnology, Inc.

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    The activation of <t>EFGR</t> by the treatment with bile acids in GS4.1 cells. Confluent GS4.1 cells were treated with mock medium, CDCA, GCDCA, or UDCA for 30 min, and then cell lysates were prepared and Western blot analysis detecting <t>phosphor-EGFR</t> or EGFR.
    Egfr, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CRISPR Cas9 KO Plasmids consists of EGFR specific 20 nt guide RNA sequences derived from the GeCKO v2 library For CRISPR gene knockout gRNA sequences direct the Cas9 protein to
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    The activation of EFGR by the treatment with bile acids in GS4.1 cells. Confluent GS4.1 cells were treated with mock medium, CDCA, GCDCA, or UDCA for 30 min, and then cell lysates were prepared and Western blot analysis detecting phosphor-EGFR or EGFR.

    Journal: Intervirology

    Article Title: Bile Acids Promote HCV Replication through the EGFR/ERK Pathway in Replicon-Harboring Cells

    doi: 10.1159/000321452

    Figure Lengend Snippet: The activation of EFGR by the treatment with bile acids in GS4.1 cells. Confluent GS4.1 cells were treated with mock medium, CDCA, GCDCA, or UDCA for 30 min, and then cell lysates were prepared and Western blot analysis detecting phosphor-EGFR or EGFR.

    Article Snippet: Antibodies against HCV NS5b, β-actin, phosphor-EFGR or EGFR were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif., USA).

    Techniques: Activation Assay, Western Blot

    Inhibitors of EGFR or ERK in bile acid-mediated promotion of HCV replication. Semiconfluent GS4.1 or 1A7 cells were incubated with mock medium, CDCA, AG1478 or CDCA + AG1478 for 24 h, and HCV replication was measured by detecting HCV RNA ( a, b ) or protein

    Journal: Intervirology

    Article Title: Bile Acids Promote HCV Replication through the EGFR/ERK Pathway in Replicon-Harboring Cells

    doi: 10.1159/000321452

    Figure Lengend Snippet: Inhibitors of EGFR or ERK in bile acid-mediated promotion of HCV replication. Semiconfluent GS4.1 or 1A7 cells were incubated with mock medium, CDCA, AG1478 or CDCA + AG1478 for 24 h, and HCV replication was measured by detecting HCV RNA ( a, b ) or protein

    Article Snippet: Antibodies against HCV NS5b, β-actin, phosphor-EFGR or EGFR were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif., USA).

    Techniques: Incubation

    Inhibitors of EGFR or ERK in anti-HCV action of IFN in the presence of CDCA. HCV RNA levels were measured after 24 h treatments with IFN 5 U/ml (IFN5), CDCA 100 μM (CDCA100) + IFN5, or CDCA100 + IFN5 + (AG1478 30 μM [AG30] or U0126 20

    Journal: Intervirology

    Article Title: Bile Acids Promote HCV Replication through the EGFR/ERK Pathway in Replicon-Harboring Cells

    doi: 10.1159/000321452

    Figure Lengend Snippet: Inhibitors of EGFR or ERK in anti-HCV action of IFN in the presence of CDCA. HCV RNA levels were measured after 24 h treatments with IFN 5 U/ml (IFN5), CDCA 100 μM (CDCA100) + IFN5, or CDCA100 + IFN5 + (AG1478 30 μM [AG30] or U0126 20

    Article Snippet: Antibodies against HCV NS5b, β-actin, phosphor-EFGR or EGFR were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif., USA).

    Techniques:

    Tumorigenic potential of low passage mucoepidermoid carcinoma cells sorted for ALDH/CD44 A., B. Graphs depicting tumor volume of A. UM-HMC-3A or B. UM-HMC-3B xenograft cells FACS-sorted for ALDH/CD44. Scaffolds were seeded with either 400 ALDH high CD44 high or 4,000 ALDH low CD44 low cells and transplanted into the subcutaneous space of SCID mice. Existing tumors were retrieved, re-sorted and 400 ALDH high CD44 high or 4,000 ALDH low CD44 low cells seeded into new scaffolds, and serially passaged in vivo . C. Table depicting the number of tumors grown in the ALDH high CD44 high versus ALDH low CD44 low populations for each passage performed. D. H E staining of tumors generated with FACS-sorted ALDH high CD44 high and ALDH low CD44 low cells. Images were taken at 100X. E. UM-HMC-3A and UM-HMC-3B cells were sorted for ALDH high CD44 high or combined ALDH high CD44 low , ALDH low CD44 high , and ALDH low CD44 low (non-CSC population). NP-40 lysis buffer was used to prepare whole cell lysates that were resolved using PAGE. Membranes were probed using antibodies a 1:1000 dilution against human mTor, p-mTor, Akt, p-Akt, S6K, p-S6K, p-EGFR; 1:2000 dilution of EGFR, and beta-actin.

    Journal: Oncotarget

    Article Title: ALDH/CD44 identifies uniquely tumorigenic cancer stem cells in salivary gland mucoepidermoid carcinomas

    doi:

    Figure Lengend Snippet: Tumorigenic potential of low passage mucoepidermoid carcinoma cells sorted for ALDH/CD44 A., B. Graphs depicting tumor volume of A. UM-HMC-3A or B. UM-HMC-3B xenograft cells FACS-sorted for ALDH/CD44. Scaffolds were seeded with either 400 ALDH high CD44 high or 4,000 ALDH low CD44 low cells and transplanted into the subcutaneous space of SCID mice. Existing tumors were retrieved, re-sorted and 400 ALDH high CD44 high or 4,000 ALDH low CD44 low cells seeded into new scaffolds, and serially passaged in vivo . C. Table depicting the number of tumors grown in the ALDH high CD44 high versus ALDH low CD44 low populations for each passage performed. D. H E staining of tumors generated with FACS-sorted ALDH high CD44 high and ALDH low CD44 low cells. Images were taken at 100X. E. UM-HMC-3A and UM-HMC-3B cells were sorted for ALDH high CD44 high or combined ALDH high CD44 low , ALDH low CD44 high , and ALDH low CD44 low (non-CSC population). NP-40 lysis buffer was used to prepare whole cell lysates that were resolved using PAGE. Membranes were probed using antibodies a 1:1000 dilution against human mTor, p-mTor, Akt, p-Akt, S6K, p-S6K, p-EGFR; 1:2000 dilution of EGFR, and beta-actin.

    Article Snippet: Membranes were probed using antibodies a 1:1000 dilution against human mTor, p-mTor, Akt, p-Akt, S6K, p-S6K (Cell Signaling; Beverly, MA, USA); 1:2000 dilution of EGFR, a 1:1000 dilution of p-EGFR, and beta-actin (Santa Cruz Biotechnology; Santa Cruz, CA, USA) overnight at 4°C.

    Techniques: FACS, Mouse Assay, In Vivo, Staining, Generated, Lysis, Polyacrylamide Gel Electrophoresis

    Topo I promotes EGFR gene expression at the mRNA level in both transformed and nontransformed cells. (A) JNK pathway stimulates EGFR mRNA expression. HT-1080 cells were treated with chemical JNK inhibitor SP600125 for 3 and 6 h, and the EGFR mRNA expression

    Journal: Molecular and Cellular Biology

    Article Title: DNA Topoisomerase I Is a Cofactor for c-Jun in the Regulation of Epidermal Growth Factor Receptor Expression and Cancer Cell Proliferation

    doi: 10.1128/MCB.25.12.5040-5051.2005

    Figure Lengend Snippet: Topo I promotes EGFR gene expression at the mRNA level in both transformed and nontransformed cells. (A) JNK pathway stimulates EGFR mRNA expression. HT-1080 cells were treated with chemical JNK inhibitor SP600125 for 3 and 6 h, and the EGFR mRNA expression

    Article Snippet: Antibodies to Topo I, c-Jun, TBP, cyclin D1, cyclin E, JNK2, p-JNK, EGFR, His, hemagglutinin (HA), glutathione S -transferase (GST), CDK4, PARP, and PCNA were purchased from Santa Cruz Biotechnology.

    Techniques: Expressing, Transformation Assay

    Tβ4 treatment induces EGFR phosphorylation/activation and the expression of MBP, Mig-6 and miR-200

    Journal: Journal of neurochemistry

    Article Title: Thymosin beta 4 up-regulates miR-200a expression and induces differentiation and survival of rat brain progenitor cells

    doi: 10.1111/jnc.13394

    Figure Lengend Snippet: Tβ4 treatment induces EGFR phosphorylation/activation and the expression of MBP, Mig-6 and miR-200

    Article Snippet: For Western blot analysis, rabbit antiserum for MBP (1:200; Dako, Carpinteria, CA, USA), monoclonal antibodies (1:500) for EGFR and phosphorylated EGFR; monoclonal antibodies (1:1000) for phosphorylated ERK1, c-JUN and FOG-2 (Santa Cruz Biotechnology, Dallas, Texas, USA); rabbit polyclonal antibodies (1:500) for Grb2, Mig-6, Pten, p53 (Cell Signaling Technology, Danver, MA, USA) and mouse monoclonal α-tubulin antibodies (1:5000; Sigma, St Louis, Mo) were used.

    Techniques: Activation Assay, Expressing

    Transfection with miR-200b-a-429 cluster enhances EGFR signaling and MBP synthesis, and inhibits Grb2 synthesis and ERK1phosphorylation

    Journal: Journal of neurochemistry

    Article Title: Thymosin beta 4 up-regulates miR-200a expression and induces differentiation and survival of rat brain progenitor cells

    doi: 10.1111/jnc.13394

    Figure Lengend Snippet: Transfection with miR-200b-a-429 cluster enhances EGFR signaling and MBP synthesis, and inhibits Grb2 synthesis and ERK1phosphorylation

    Article Snippet: For Western blot analysis, rabbit antiserum for MBP (1:200; Dako, Carpinteria, CA, USA), monoclonal antibodies (1:500) for EGFR and phosphorylated EGFR; monoclonal antibodies (1:1000) for phosphorylated ERK1, c-JUN and FOG-2 (Santa Cruz Biotechnology, Dallas, Texas, USA); rabbit polyclonal antibodies (1:500) for Grb2, Mig-6, Pten, p53 (Cell Signaling Technology, Danver, MA, USA) and mouse monoclonal α-tubulin antibodies (1:5000; Sigma, St Louis, Mo) were used.

    Techniques: Transfection