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    Name:
    EGFR
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    Catalog Number:
    srp5023
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    Structured Review

    Millipore egfr
    EGFR

    https://www.bioz.com/result/egfr/product/Millipore
    Average 96 stars, based on 103 article reviews
    Price from $9.99 to $1999.99
    egfr - by Bioz Stars, 2020-11
    96/100 stars

    Images

    1) Product Images from "89Zr-Onartuzumab PET imaging of c-MET receptor dynamics"

    Article Title: 89Zr-Onartuzumab PET imaging of c-MET receptor dynamics

    Journal: European Journal of Nuclear Medicine and Molecular Imaging

    doi: 10.1007/s00259-017-3672-x

    a In vitro flow cytometric analysis of EGFR and c-MET membrane expression in HCC827ErlRes cells normalized to expression in parental cell line HCC827. b In vitro MTT proliferation assay in HCC827 and HCC827ErlRes cells with exposure to increasing concentrations of erlotinib for 4 days. c In vitro flow cytometric analysis of EGFR and c-MET membrane expression in HCC827 and HCC827ErlRes cells after 24 h treatment with 25, 50 and 100 nM NVP-AUY-922 normalized to untreated controls. d In vitro MTT proliferation assay in HCC827 and HCC827ErlRes cells with exposure to increasing concentrations of NVP-AUY-922 for 4 days
    Figure Legend Snippet: a In vitro flow cytometric analysis of EGFR and c-MET membrane expression in HCC827ErlRes cells normalized to expression in parental cell line HCC827. b In vitro MTT proliferation assay in HCC827 and HCC827ErlRes cells with exposure to increasing concentrations of erlotinib for 4 days. c In vitro flow cytometric analysis of EGFR and c-MET membrane expression in HCC827 and HCC827ErlRes cells after 24 h treatment with 25, 50 and 100 nM NVP-AUY-922 normalized to untreated controls. d In vitro MTT proliferation assay in HCC827 and HCC827ErlRes cells with exposure to increasing concentrations of NVP-AUY-922 for 4 days

    Techniques Used: In Vitro, Flow Cytometry, Expressing, MTT Assay, Proliferation Assay

    2) Product Images from "Epidermal Growth Factor Receptor Activation by Epidermal Growth Factor Mediates Oxidant-Induced Goblet Cell Metaplasia in Human Airway Epithelium"

    Article Title: Epidermal Growth Factor Receptor Activation by Epidermal Growth Factor Mediates Oxidant-Induced Goblet Cell Metaplasia in Human Airway Epithelium

    Journal: American Journal of Respiratory Cell and Molecular Biology

    doi: 10.1165/rcmb.2005-0386OC

    ROS-induce Bcl-2 mRNA expression. Human NHBE cells were exposed to PBS or X/XO for three consecutive days in the presence or absence of catalase, PI, GM6001, or functionally blocking anti-EGFR monoclonal antibodies (RAb). Bcl-2 mRNA expression assessed
    Figure Legend Snippet: ROS-induce Bcl-2 mRNA expression. Human NHBE cells were exposed to PBS or X/XO for three consecutive days in the presence or absence of catalase, PI, GM6001, or functionally blocking anti-EGFR monoclonal antibodies (RAb). Bcl-2 mRNA expression assessed

    Techniques Used: Expressing, Blocking Assay

    3) Product Images from "HER2 signaling regulates HER2 localization and membrane retention"

    Article Title: HER2 signaling regulates HER2 localization and membrane retention

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0174849

    HER2 signaling prevents HER2 internalization. A) Con-focal images of immunofluorescence for HER2 (green) and EGFR (red) in control (top row) and HER2KD-SKBR3 (bottom row) cells at baseline in serum-containing media. White arrow points to internalized HER2. Exposure times are longer in HER2KD cells to compensate for reduced HER2 levels. B) Con-focal images of immunofluorescence for HER2 (green) and EGFR (red) in control (top row) and HER2KD-SKBR3 (bottom row) cells exposed EGF for 2 hrs. Panels on right represent magnifications of boxed areas. White arrow indicates internalized HER2 and EGFR. C) Quantification of cell surface HER2 as detected by isolation of cell-surface biotinylated proteins. Each bar represents the mean ± SEM of 3 separate experiments in cells at baseline and after EGF treatment. Asterisk denotes p
    Figure Legend Snippet: HER2 signaling prevents HER2 internalization. A) Con-focal images of immunofluorescence for HER2 (green) and EGFR (red) in control (top row) and HER2KD-SKBR3 (bottom row) cells at baseline in serum-containing media. White arrow points to internalized HER2. Exposure times are longer in HER2KD cells to compensate for reduced HER2 levels. B) Con-focal images of immunofluorescence for HER2 (green) and EGFR (red) in control (top row) and HER2KD-SKBR3 (bottom row) cells exposed EGF for 2 hrs. Panels on right represent magnifications of boxed areas. White arrow indicates internalized HER2 and EGFR. C) Quantification of cell surface HER2 as detected by isolation of cell-surface biotinylated proteins. Each bar represents the mean ± SEM of 3 separate experiments in cells at baseline and after EGF treatment. Asterisk denotes p

    Techniques Used: Immunofluorescence, Isolation

    4) Product Images from "The ErbB2ΔEx16 splice variant is a major oncogenic driver in breast cancer that promotes a pro-metastatic tumor microenvironment"

    Article Title: The ErbB2ΔEx16 splice variant is a major oncogenic driver in breast cancer that promotes a pro-metastatic tumor microenvironment

    Journal: Oncogene

    doi: 10.1038/onc.2016.129

    Immunoblot analysis of ErbB2-expressing tumors. (A) Full length ErbB2-driven tumors co-express heterodimer partners ErbB3 and EGFR, whereas ErbB2ΔEx16 tumors arise in the absence of heterodimerization partners. (B) ErbB2ΔEx16 displays heightened activation of Src-family kinases (SFK) and p38 MAPK. (C) Additionally, ErbB2ΔEx16-expressing tumors preferentially activate a distinct subset of transcription factors, primarily Smad2, HIF1α, Stat3, and YB1. (D) Unsupervised hierarchical clustering of tumors results in grouping of tumors by genotype.
    Figure Legend Snippet: Immunoblot analysis of ErbB2-expressing tumors. (A) Full length ErbB2-driven tumors co-express heterodimer partners ErbB3 and EGFR, whereas ErbB2ΔEx16 tumors arise in the absence of heterodimerization partners. (B) ErbB2ΔEx16 displays heightened activation of Src-family kinases (SFK) and p38 MAPK. (C) Additionally, ErbB2ΔEx16-expressing tumors preferentially activate a distinct subset of transcription factors, primarily Smad2, HIF1α, Stat3, and YB1. (D) Unsupervised hierarchical clustering of tumors results in grouping of tumors by genotype.

    Techniques Used: Expressing, Activation Assay

    5) Product Images from "DDX3X Induces Primary EGFR-TKI Resistance Based on Intratumor Heterogeneity in Lung Cancer Cells Harboring EGFR-Activating Mutations"

    Article Title: DDX3X Induces Primary EGFR-TKI Resistance Based on Intratumor Heterogeneity in Lung Cancer Cells Harboring EGFR-Activating Mutations

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111019

    A. Immunoblotting analysis of EGFR, phospho-EGFR (Tyr1068), and DDX3X in cancer cells without EGF supplementation. B. Immunoblotting analysis of phospho-EGFR at Tyr1068, Tyr1173, and Tyr845 in parental PC9 cells and A-1 cells. C. Kinetic analysis of EGFR signaling in parental PC9 cells and A-1 cells. EGFR, phospho-EGFR (Tyr1068), Akt, phospho-Akt, ERK1/2, and phospho-ERK1/2 levels were analyzed by immunoblotting at 0, 15, 30, 60, 120, and 900 min after the addition of 100 ng/mL EGF.
    Figure Legend Snippet: A. Immunoblotting analysis of EGFR, phospho-EGFR (Tyr1068), and DDX3X in cancer cells without EGF supplementation. B. Immunoblotting analysis of phospho-EGFR at Tyr1068, Tyr1173, and Tyr845 in parental PC9 cells and A-1 cells. C. Kinetic analysis of EGFR signaling in parental PC9 cells and A-1 cells. EGFR, phospho-EGFR (Tyr1068), Akt, phospho-Akt, ERK1/2, and phospho-ERK1/2 levels were analyzed by immunoblotting at 0, 15, 30, 60, 120, and 900 min after the addition of 100 ng/mL EGF.

    Techniques Used:

    6) Product Images from "NF-κB-induced KIAA1199 promotes survival through EGFR signalling"

    Article Title: NF-κB-induced KIAA1199 promotes survival through EGFR signalling

    Journal: Nature Communications

    doi: 10.1038/ncomms6232

    EGF-dependent signalling requires KIAA1199 in breast cancer-derived cells. ( a , b ) Impaired expression of ErbB members and defective EGFR signalling on KIAA1199 deficiency in breast cancer-derived cells. On the left, serum-starved control or KIAA1199-deficient MCF7 ( a ) or SK-BR-3 ( b ) cells were untreated or stimulated with EGF (100 ng ml −1 ) for the indicated periods of time and the resulting cell extracts were subjected to WB analyses using the indicated antibodies. On the right, total RNAs from control or KIAA1199-depleted (shRNA KIAA1199#2) CaSki cells were subjected to real-time PCR to assess KIAA1199 mRNA levels. The abundance of transcripts in control cells was set to 1 and their levels in KIAA1199-depleted cells were relative to that after normalization with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ( c ) KIAA1199 promotes EGF-dependent signalling in immortalized breast epithelial cells. Serum-starved control or KIAA1199-depleted NMuMG cells were serum starved and were untreated or stimulated with EGF (100 ng ml −1 ) for the indicated periods of time. WB analyses were conducted with the resulting cell extracts.
    Figure Legend Snippet: EGF-dependent signalling requires KIAA1199 in breast cancer-derived cells. ( a , b ) Impaired expression of ErbB members and defective EGFR signalling on KIAA1199 deficiency in breast cancer-derived cells. On the left, serum-starved control or KIAA1199-deficient MCF7 ( a ) or SK-BR-3 ( b ) cells were untreated or stimulated with EGF (100 ng ml −1 ) for the indicated periods of time and the resulting cell extracts were subjected to WB analyses using the indicated antibodies. On the right, total RNAs from control or KIAA1199-depleted (shRNA KIAA1199#2) CaSki cells were subjected to real-time PCR to assess KIAA1199 mRNA levels. The abundance of transcripts in control cells was set to 1 and their levels in KIAA1199-depleted cells were relative to that after normalization with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ( c ) KIAA1199 promotes EGF-dependent signalling in immortalized breast epithelial cells. Serum-starved control or KIAA1199-depleted NMuMG cells were serum starved and were untreated or stimulated with EGF (100 ng ml −1 ) for the indicated periods of time. WB analyses were conducted with the resulting cell extracts.

    Techniques Used: Derivative Assay, Expressing, Western Blot, shRNA, Real-time Polymerase Chain Reaction

    KIAA1199 limits EGF-dependent EGFR degradation through lysosomes. ( a ) KIAA1199 deficiency enhances EGFR co-localization with lysosomes on EGF stimulation. Control or KIAA1199-deficient CaSki cells were serum starved and subsequently untreated or stimulated with EGF for 15 or 30 min and subjected to immunofluorescence (IF) analyses. EGF-bound to EGFR as well as LAMP2-positive lysosomes were detected. Arrows indicates examples of EGF-bound EGFR that co-localizes with LAMP2-positive lysosomes. On the right, a quantification of co-localization of EGF-bound EGFR with LAMP2-positive lysosomes per cell on 15 min of EGF stimulation is illustrated. Values obtained in control cells was set to 1 and values obtained in other experimental conditions were relative to that. Data from one representative experiment performed in triplicates (means±s.d.) are shown (Student’s t -test, P -values: ***
    Figure Legend Snippet: KIAA1199 limits EGF-dependent EGFR degradation through lysosomes. ( a ) KIAA1199 deficiency enhances EGFR co-localization with lysosomes on EGF stimulation. Control or KIAA1199-deficient CaSki cells were serum starved and subsequently untreated or stimulated with EGF for 15 or 30 min and subjected to immunofluorescence (IF) analyses. EGF-bound to EGFR as well as LAMP2-positive lysosomes were detected. Arrows indicates examples of EGF-bound EGFR that co-localizes with LAMP2-positive lysosomes. On the right, a quantification of co-localization of EGF-bound EGFR with LAMP2-positive lysosomes per cell on 15 min of EGF stimulation is illustrated. Values obtained in control cells was set to 1 and values obtained in other experimental conditions were relative to that. Data from one representative experiment performed in triplicates (means±s.d.) are shown (Student’s t -test, P -values: ***

    Techniques Used: Immunofluorescence

    KIAA1199 connects Semaphorin 3A signalling to EGFR phosphorylation. ( a ) Semaphorin 3A-mediated EGFR phosphorylation requires KIAA1199. Control or KIAA1199-depleted CaSki cells were untreated or stimulated with Semaphorin 3A (100 ng ml −1 ) and WB analyses were carried out on the resulting cell extracts (lysis in SDS 1%). ( b ) KIAA1199 deficiency does not have an impact on EGFR mRNA levels in cervical cancer cells. Total RNAs from control or KIAA1199-depleted (shRNA KIAA1199#1 or shRNA KIAA1199#2) CaSki cells were subjected to real-time PCR, to assess EGFR mRNA levels. The abundance of transcripts in control cells was set to 1 and their levels in KIAA1199-depleted cells were relative to that after normalization with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data from two independent experiments (means±s.d.) are shown. ( c ) Plexin A2 deficiency potentiates Semaphorin 3A-mediated EGFR phosphorylation. Control or Plexin A2-depleted CaSki cells were left untreated or stimulated with Semaphorin 3A for the indicated periods of time. The resulting cell extracts (lysis in SDS 1%) were subjected to WBs using the indicated antibodies. ( d ) Plexin A2 deficiency prolongs the binding of KIAA1199 to EGFR on EGF stimulation. Control or Plexin A2-deficient cells were untreated or stimulated with EGF for the indicated periods of time. Cell extracts were subjected to anti-FLAG (negative control) or -EGFR immunoprecipitations followed by anti- KIAA1199 or -EGFR WBs (top panels). Crude cell extracts were subjected to anti-pEGFR (Y845 and Y1068) (to validate the triggering of the EGF-dependent pathway), -EGFR, -KIAA1199, -Plexin A2 and -HSP90 WBs, as indicated. ( e ) KIAA1199 binds EGFR through its N-terminal domain. Cells were transfected with the indicated expression plasmids and protein extracts were subjected to anti-HA (negative control) or -FLAG IPs followed by an anti-EGFR WB (top panel). Crude cell extracts were also subjected to anti-EGFR and -FLAG WB analyses (bottom panels).
    Figure Legend Snippet: KIAA1199 connects Semaphorin 3A signalling to EGFR phosphorylation. ( a ) Semaphorin 3A-mediated EGFR phosphorylation requires KIAA1199. Control or KIAA1199-depleted CaSki cells were untreated or stimulated with Semaphorin 3A (100 ng ml −1 ) and WB analyses were carried out on the resulting cell extracts (lysis in SDS 1%). ( b ) KIAA1199 deficiency does not have an impact on EGFR mRNA levels in cervical cancer cells. Total RNAs from control or KIAA1199-depleted (shRNA KIAA1199#1 or shRNA KIAA1199#2) CaSki cells were subjected to real-time PCR, to assess EGFR mRNA levels. The abundance of transcripts in control cells was set to 1 and their levels in KIAA1199-depleted cells were relative to that after normalization with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data from two independent experiments (means±s.d.) are shown. ( c ) Plexin A2 deficiency potentiates Semaphorin 3A-mediated EGFR phosphorylation. Control or Plexin A2-depleted CaSki cells were left untreated or stimulated with Semaphorin 3A for the indicated periods of time. The resulting cell extracts (lysis in SDS 1%) were subjected to WBs using the indicated antibodies. ( d ) Plexin A2 deficiency prolongs the binding of KIAA1199 to EGFR on EGF stimulation. Control or Plexin A2-deficient cells were untreated or stimulated with EGF for the indicated periods of time. Cell extracts were subjected to anti-FLAG (negative control) or -EGFR immunoprecipitations followed by anti- KIAA1199 or -EGFR WBs (top panels). Crude cell extracts were subjected to anti-pEGFR (Y845 and Y1068) (to validate the triggering of the EGF-dependent pathway), -EGFR, -KIAA1199, -Plexin A2 and -HSP90 WBs, as indicated. ( e ) KIAA1199 binds EGFR through its N-terminal domain. Cells were transfected with the indicated expression plasmids and protein extracts were subjected to anti-HA (negative control) or -FLAG IPs followed by an anti-EGFR WB (top panel). Crude cell extracts were also subjected to anti-EGFR and -FLAG WB analyses (bottom panels).

    Techniques Used: Western Blot, Lysis, shRNA, Real-time Polymerase Chain Reaction, Binding Assay, Negative Control, Transfection, Expressing

    KIAA1199 promotes EGF-dependent signalling. ( a ) Defective EGF-dependent signalling on KIAA1199 deficiency in cervical cancer cells. Serum-starved control or KIAA1199-depleted CaSki cells (shRNA KIAA1199#2) were untreated or stimulated with EGF (100 ng ml −1 ) from 5 to 30 min. The resulting cell extracts were subjected to anti-FLAG (negative control) or -EGFR IPs followed by anti-Src, -c-Cbl and -EGFR WBs (top three panels). Crude cell extracts were also subjected to WB analyses using the indicated antibodies. ( b ) Optimal EGF-dependent Ras activation requires KIAA1199 in CaSki cells. Ras activity on stimulation with EGF (10 ng ml −1 ) was assessed in control or KIAA1199-depleted cells. Ras + and His-Ras were used as positive controls. An anti-pan-RAS WB was carried out for normalization purposes (‘input’, bottom panel). ( c ) Defective HER2 expression on KIAA1199 deficiency in cervical cancer cells. Extracts from control or KIAA1199-depleted CaSki cells were subjected to WB analyses using the indicated antibodies. ( d ) Defective MEK1 activation on stimulation by NRG-1 in KIAA1199-deficient cervical cancer cells. Serum-starved control or KIAA1199-deficient CaSki cells were untreated or stimulated with NRG1 (100 ng ml −1 ) for the indicated periods of time and the resulting cell extracts were subjected to WB analyses, using the indicated antibodies. ( e ) KIAA1199 is dispensable for EGFR dimer formation on EGF stimulation in cervical cancer-derived cells. Serum-starved CaSki cells were unstimulated or treated with EGF (100 ng ml −1 ) for the indicated periods of time. Cells were subsequently cross-linked using 1 mM of bis(sulfosuccinimidyl) suberate (BS 3 ) as chemical cross-linker. On the left, extracts were subjected to anti -EGFR WB analyses to detect both EGFR monomers and dimers. On the right, quantification of the dimer/monomer/HSP90 ratio in both control and KIAA1199-depleted cells subjected to EGF stimulations or not. The value of the dimer/monomer/HSP90 ratio in control and unstimulated cells was set to 1 and values obtained in other experimental conditions were relative to that. Data from three independent experiments (means±s.d.) are shown. ( f ) KIAA1199 differentially controls the expression of ErbB ligands in CaSki cells. Total RNAs from control or KIAA1199-depleted (shRNA KIAA1199#1 or shRNA KIAA1199#2) CaSki cells were subjected to real-time PCR to assess mRNA levels of the indicated ErbB ligands. The abundance of transcripts in control cells was set to 1 and their levels in KIAA1199-depleted cells were relative to that after normalization with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data from three independent experiments performed in triplicates (means±s.d.) are shown (Student’s t -test, P -values: ***
    Figure Legend Snippet: KIAA1199 promotes EGF-dependent signalling. ( a ) Defective EGF-dependent signalling on KIAA1199 deficiency in cervical cancer cells. Serum-starved control or KIAA1199-depleted CaSki cells (shRNA KIAA1199#2) were untreated or stimulated with EGF (100 ng ml −1 ) from 5 to 30 min. The resulting cell extracts were subjected to anti-FLAG (negative control) or -EGFR IPs followed by anti-Src, -c-Cbl and -EGFR WBs (top three panels). Crude cell extracts were also subjected to WB analyses using the indicated antibodies. ( b ) Optimal EGF-dependent Ras activation requires KIAA1199 in CaSki cells. Ras activity on stimulation with EGF (10 ng ml −1 ) was assessed in control or KIAA1199-depleted cells. Ras + and His-Ras were used as positive controls. An anti-pan-RAS WB was carried out for normalization purposes (‘input’, bottom panel). ( c ) Defective HER2 expression on KIAA1199 deficiency in cervical cancer cells. Extracts from control or KIAA1199-depleted CaSki cells were subjected to WB analyses using the indicated antibodies. ( d ) Defective MEK1 activation on stimulation by NRG-1 in KIAA1199-deficient cervical cancer cells. Serum-starved control or KIAA1199-deficient CaSki cells were untreated or stimulated with NRG1 (100 ng ml −1 ) for the indicated periods of time and the resulting cell extracts were subjected to WB analyses, using the indicated antibodies. ( e ) KIAA1199 is dispensable for EGFR dimer formation on EGF stimulation in cervical cancer-derived cells. Serum-starved CaSki cells were unstimulated or treated with EGF (100 ng ml −1 ) for the indicated periods of time. Cells were subsequently cross-linked using 1 mM of bis(sulfosuccinimidyl) suberate (BS 3 ) as chemical cross-linker. On the left, extracts were subjected to anti -EGFR WB analyses to detect both EGFR monomers and dimers. On the right, quantification of the dimer/monomer/HSP90 ratio in both control and KIAA1199-depleted cells subjected to EGF stimulations or not. The value of the dimer/monomer/HSP90 ratio in control and unstimulated cells was set to 1 and values obtained in other experimental conditions were relative to that. Data from three independent experiments (means±s.d.) are shown. ( f ) KIAA1199 differentially controls the expression of ErbB ligands in CaSki cells. Total RNAs from control or KIAA1199-depleted (shRNA KIAA1199#1 or shRNA KIAA1199#2) CaSki cells were subjected to real-time PCR to assess mRNA levels of the indicated ErbB ligands. The abundance of transcripts in control cells was set to 1 and their levels in KIAA1199-depleted cells were relative to that after normalization with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data from three independent experiments performed in triplicates (means±s.d.) are shown (Student’s t -test, P -values: ***

    Techniques Used: shRNA, Negative Control, Western Blot, Activation Assay, Activity Assay, Expressing, Derivative Assay, Real-time Polymerase Chain Reaction

    7) Product Images from "DeltaNp63alpha-Mediated Induction of Epidermal Growth Factor Receptor Promotes Pancreatic Cancer Cell Growth and Chemoresistance"

    Article Title: DeltaNp63alpha-Mediated Induction of Epidermal Growth Factor Receptor Promotes Pancreatic Cancer Cell Growth and Chemoresistance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026815

    A distinct role of ΔNp63α in HEK293 and H1299 cells. A , ΔNp63α does not affect EGFR or 14-3-3σ protein levels in HEK293 or H1299 cells. Representative blot of three independent experiments is shown. B , ΔNp63α fails to upregulate EGFR promoter but modulates 14-3-3σ promoter in a luciferase reporter assay. Cells were transfected with wildtype EGFR or 14-3-3σ promoter luciferase construct as indicated, along with pCMVβ plasmid, with or without wildtype p53, ΔNp63α, ΔNp63α DBDmut , or TAp63α. At 48–72 h after transfection, the luciferase activity was determined. The transfection efficiency was standardized against β-galactosidase activity. Results are indicative of three independent experiments performed in duplicates. C , ΔNp63α increases 14-3-3σ mRNA transcript levels in H1299 cells. H1299 cells were transfected with ΔNp63α, ΔNp63α DBDmut or vector control. At 36 hours total RNA was isolated, reverse-transcribed and subjected to real-time PCR with probe specific for 14-3-3σ. Results were normalized to 18S levels. Data are the mean of two independent experiments done in duplicates in which similar results were obtained. D , ΔNp63α does not enhance anchorage-independent growth, migration or invasion in H1299 cells. H1299 cells were transfected with ΔNp63α or vector control. Soft agar, transwell migration and invasion assays were performed as described above. *, p
    Figure Legend Snippet: A distinct role of ΔNp63α in HEK293 and H1299 cells. A , ΔNp63α does not affect EGFR or 14-3-3σ protein levels in HEK293 or H1299 cells. Representative blot of three independent experiments is shown. B , ΔNp63α fails to upregulate EGFR promoter but modulates 14-3-3σ promoter in a luciferase reporter assay. Cells were transfected with wildtype EGFR or 14-3-3σ promoter luciferase construct as indicated, along with pCMVβ plasmid, with or without wildtype p53, ΔNp63α, ΔNp63α DBDmut , or TAp63α. At 48–72 h after transfection, the luciferase activity was determined. The transfection efficiency was standardized against β-galactosidase activity. Results are indicative of three independent experiments performed in duplicates. C , ΔNp63α increases 14-3-3σ mRNA transcript levels in H1299 cells. H1299 cells were transfected with ΔNp63α, ΔNp63α DBDmut or vector control. At 36 hours total RNA was isolated, reverse-transcribed and subjected to real-time PCR with probe specific for 14-3-3σ. Results were normalized to 18S levels. Data are the mean of two independent experiments done in duplicates in which similar results were obtained. D , ΔNp63α does not enhance anchorage-independent growth, migration or invasion in H1299 cells. H1299 cells were transfected with ΔNp63α or vector control. Soft agar, transwell migration and invasion assays were performed as described above. *, p

    Techniques Used: Luciferase, Reporter Assay, Transfection, Construct, Plasmid Preparation, Activity Assay, Isolation, Real-time Polymerase Chain Reaction, Migration

    ΔNp63α is a transcriptional activator of EGFR and 14-3-3σ in pancreatic cancer cells. A , ΔNp63α DBDmut does not upregulate 14-3-3σ. B and C , ΔNp63α upregulates EGFR and 14-3-3σ promoter in a luciferase assay. PANC-1 cells were transfected with wildtype EGFR, mutant EGFR or 14-3-3σ promoter luciferase construct as indicated, along with pCMVβ plasmid, with or without wildtype p53, ΔNp63α, ΔNp63α DBDmut , or TAp63α. At 48–72 h after transfection, the luciferase activity was determined. The transfection efficiency was standardized against β-galactosidase activity. Results are indicative of four independent experiments. *, p
    Figure Legend Snippet: ΔNp63α is a transcriptional activator of EGFR and 14-3-3σ in pancreatic cancer cells. A , ΔNp63α DBDmut does not upregulate 14-3-3σ. B and C , ΔNp63α upregulates EGFR and 14-3-3σ promoter in a luciferase assay. PANC-1 cells were transfected with wildtype EGFR, mutant EGFR or 14-3-3σ promoter luciferase construct as indicated, along with pCMVβ plasmid, with or without wildtype p53, ΔNp63α, ΔNp63α DBDmut , or TAp63α. At 48–72 h after transfection, the luciferase activity was determined. The transfection efficiency was standardized against β-galactosidase activity. Results are indicative of four independent experiments. *, p

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

    8) Product Images from "ATP-mediated Activation of the NADPH Oxidase DUOX1 Mediates Airway Epithelial Responses to Bacterial Stimuli *"

    Article Title: ATP-mediated Activation of the NADPH Oxidase DUOX1 Mediates Airway Epithelial Responses to Bacterial Stimuli *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M809761200

    ATP-mediated DUOX1 activation promotes TGF-α shedding in HBE1 cells. A, HBE1 cells were stimulated with either ATP or α-ASGM1 for 2 h, in the absence or presence of α-EGFR mAb (225; 4 μg/ml), and conditioned media were
    Figure Legend Snippet: ATP-mediated DUOX1 activation promotes TGF-α shedding in HBE1 cells. A, HBE1 cells were stimulated with either ATP or α-ASGM1 for 2 h, in the absence or presence of α-EGFR mAb (225; 4 μg/ml), and conditioned media were

    Techniques Used: Activation Assay

    Schematic representation of DUOX1 involvement in TLR-mediated IL-8 production in response to bacterial stimuli. Cell stimulation with LPS or α-ASGM1 mediates release of ATP, which activates EGFR-ERK1/2-NF-κB signaling cascades which is
    Figure Legend Snippet: Schematic representation of DUOX1 involvement in TLR-mediated IL-8 production in response to bacterial stimuli. Cell stimulation with LPS or α-ASGM1 mediates release of ATP, which activates EGFR-ERK1/2-NF-κB signaling cascades which is

    Techniques Used: Cell Stimulation

    9) Product Images from "Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells, et al. Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells"

    Article Title: Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells, et al. Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14730

    Expression level of EGFR and MET in generated variants of A375 and WM9 melanoma cell lines. A, Results of qRT‐PCR analysis of EGFR and MET expression are shown as the mean (relative expression compared to GAPDH) ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001. B, Western blotting analysis of EGFR and MET protein level in generated cell lines. Membranes were probed with antibodies directed against total EGFR and MET, and are representative for at least three independent experiments. GAPDH was used as the sample loading control
    Figure Legend Snippet: Expression level of EGFR and MET in generated variants of A375 and WM9 melanoma cell lines. A, Results of qRT‐PCR analysis of EGFR and MET expression are shown as the mean (relative expression compared to GAPDH) ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001. B, Western blotting analysis of EGFR and MET protein level in generated cell lines. Membranes were probed with antibodies directed against total EGFR and MET, and are representative for at least three independent experiments. GAPDH was used as the sample loading control

    Techniques Used: Expressing, Generated, Quantitative RT-PCR, Western Blot

    Proteolytic activity of melanoma cells with altered EGFR and MET expression. A, Representative pictures of proteolytic activity of A375 (MOCK, EGFR, shCTRL and shMET) and WM9 (shCTRL, shEGFR and shMET) cells (F‐actin visible in red) detected using FITC‐conjugated gelatin (green). Gelatin degradation indicated with white arrows is visualized as the dark areas on the fluorescently labelled gelatin background. Scale bar—10 µm. B, Quantification of digestion area calculated using ImageJ software from at least 40 cells from three independent experiments. Results are presented as the mean ± SD. C, MMP‐9 activity in concentrated conditioned media tested by gelatin zymography with (D) densitometric analysis. Results are expressed as the mean ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001
    Figure Legend Snippet: Proteolytic activity of melanoma cells with altered EGFR and MET expression. A, Representative pictures of proteolytic activity of A375 (MOCK, EGFR, shCTRL and shMET) and WM9 (shCTRL, shEGFR and shMET) cells (F‐actin visible in red) detected using FITC‐conjugated gelatin (green). Gelatin degradation indicated with white arrows is visualized as the dark areas on the fluorescently labelled gelatin background. Scale bar—10 µm. B, Quantification of digestion area calculated using ImageJ software from at least 40 cells from three independent experiments. Results are presented as the mean ± SD. C, MMP‐9 activity in concentrated conditioned media tested by gelatin zymography with (D) densitometric analysis. Results are expressed as the mean ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001

    Techniques Used: Activity Assay, Expressing, Software, Zymography

    Impact of EGFR and MET on invasion abilities and invadopodia formation in examined melanoma cell lines. A, The invasion assay performed on transwell filters coated with Matrigel for 24 h. Relative invasion factor was calculated versus control cells, where number of invading control cells is set as 100%. Results are expressed as the mean ± SD of three independent experiments. B, Representative pictures of A375 (MOCK, EGFR, shCTRL and shMET) and WM9 (shCTRL, shEGFR and shMET) cells seeded on Matrigel‐coated coverslips stained for F‐actin (red), cortactin (green) and cell nuclei (blue). Arrows indicate invadopodia. Scale bar—8 μm. C, Quantification of the average number of invadopodia in examined cells. Invadopodia formed by at least 40 cells from three independent experiments were counted, and results are presented as the mean ± SD. (**) P ≤ .01, (***) P ≤ .001
    Figure Legend Snippet: Impact of EGFR and MET on invasion abilities and invadopodia formation in examined melanoma cell lines. A, The invasion assay performed on transwell filters coated with Matrigel for 24 h. Relative invasion factor was calculated versus control cells, where number of invading control cells is set as 100%. Results are expressed as the mean ± SD of three independent experiments. B, Representative pictures of A375 (MOCK, EGFR, shCTRL and shMET) and WM9 (shCTRL, shEGFR and shMET) cells seeded on Matrigel‐coated coverslips stained for F‐actin (red), cortactin (green) and cell nuclei (blue). Arrows indicate invadopodia. Scale bar—8 μm. C, Quantification of the average number of invadopodia in examined cells. Invadopodia formed by at least 40 cells from three independent experiments were counted, and results are presented as the mean ± SD. (**) P ≤ .01, (***) P ≤ .001

    Techniques Used: Invasion Assay, Staining

    Migration abilities of melanoma cells with altered level of EGFR and MET. A, Cell trajectories and (B) migration distances of single A375 and WM9 cells analysed for 48 h using IncuCyte ® Live‐Cell Analysis System and ImageJ software. C, Representative images of wound closure, which was (D) quantified as per cent of area colonized by cells within 48 h (based on pictures analysed with an IncuCyte ® Scratch Wound Cell Migration Software Module). E, The migration assay executed on transwell filters for 24 h. Relative migration factor was calculated versus control cells, where number of migrating control cells is set as 100%. Results are expressed as the mean ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001
    Figure Legend Snippet: Migration abilities of melanoma cells with altered level of EGFR and MET. A, Cell trajectories and (B) migration distances of single A375 and WM9 cells analysed for 48 h using IncuCyte ® Live‐Cell Analysis System and ImageJ software. C, Representative images of wound closure, which was (D) quantified as per cent of area colonized by cells within 48 h (based on pictures analysed with an IncuCyte ® Scratch Wound Cell Migration Software Module). E, The migration assay executed on transwell filters for 24 h. Relative migration factor was calculated versus control cells, where number of migrating control cells is set as 100%. Results are expressed as the mean ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001

    Techniques Used: Migration, Software

    10) Product Images from "Gefitinib and Luteolin Cause Growth Arrest of Human Prostate Cancer PC-3 Cells via Inhibition of Cyclin G-Associated Kinase and Induction of miR-630"

    Article Title: Gefitinib and Luteolin Cause Growth Arrest of Human Prostate Cancer PC-3 Cells via Inhibition of Cyclin G-Associated Kinase and Induction of miR-630

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0100124

    Loss of GAK activity leads to constitutive hyper-phosphorylation of the EGFR. A, western blot analysis of EGFR expression in WT (GAK-kd +/+ ) and mutant (GAK-kd −/− ) MEFs. B, the effects of a Tyr-phosphatase inhibitor (50 mM Na 3 VO 4 ) and Ser/Thr phosphatase inhibitors (50 mM NaF and 50 mM β-glycerophosphate, or 2.5 µM okadaic acid) on the inhibition of EGFR phosphorylation by λ-phosphatase (λPPase; 200 U). A, B, the arrows indicate the phosphorylated EGFR protein. Alpha-tubulin (α-Tub) was used as a loading control. (C, D) Western blot analysis of expression levels of the EGFR and ERK1/2 in WT (+/+) and GAK-kd (−/−) MEFs following EGF stimulation for the indicated times. Cycloheximide (50 µg/ml) was added to the culture medium 1 h prior to EGF (10 µg/ml) to inhibit novel protein synthesis. The tilted and horizontal arrows indicate the phosphorylated and hyper-phosphorylated EGFR bands, respectively. GAPDH was used as a loading control. C, the arrowheads indicate differential expression of phosphorylated ERK1/2 in WT and GAK-kd MEFs. D, NT, non-treated. E, immunostaining of the EGFR protein in WT (+/+) and mutant (−/−) MEFs treated with or without the proteasome inhibitor MG132 (50 µg/ml). Notable panels are encircled by turquoise and green lines. F, the numbers of EGFR-positive cells in WT and GAK-kd (Homo) cells in the presence or absence of MG132. The data are represented as the mean ± SEM of n = 3 independent experiments at each time point.
    Figure Legend Snippet: Loss of GAK activity leads to constitutive hyper-phosphorylation of the EGFR. A, western blot analysis of EGFR expression in WT (GAK-kd +/+ ) and mutant (GAK-kd −/− ) MEFs. B, the effects of a Tyr-phosphatase inhibitor (50 mM Na 3 VO 4 ) and Ser/Thr phosphatase inhibitors (50 mM NaF and 50 mM β-glycerophosphate, or 2.5 µM okadaic acid) on the inhibition of EGFR phosphorylation by λ-phosphatase (λPPase; 200 U). A, B, the arrows indicate the phosphorylated EGFR protein. Alpha-tubulin (α-Tub) was used as a loading control. (C, D) Western blot analysis of expression levels of the EGFR and ERK1/2 in WT (+/+) and GAK-kd (−/−) MEFs following EGF stimulation for the indicated times. Cycloheximide (50 µg/ml) was added to the culture medium 1 h prior to EGF (10 µg/ml) to inhibit novel protein synthesis. The tilted and horizontal arrows indicate the phosphorylated and hyper-phosphorylated EGFR bands, respectively. GAPDH was used as a loading control. C, the arrowheads indicate differential expression of phosphorylated ERK1/2 in WT and GAK-kd MEFs. D, NT, non-treated. E, immunostaining of the EGFR protein in WT (+/+) and mutant (−/−) MEFs treated with or without the proteasome inhibitor MG132 (50 µg/ml). Notable panels are encircled by turquoise and green lines. F, the numbers of EGFR-positive cells in WT and GAK-kd (Homo) cells in the presence or absence of MG132. The data are represented as the mean ± SEM of n = 3 independent experiments at each time point.

    Techniques Used: Activity Assay, Western Blot, Expressing, Mutagenesis, Inhibition, Immunostaining

    11) Product Images from "Estrogen Receptor ? Exerts Tumor Repressive Functions in Human Malignant Pleural Mesothelioma via EGFR Inactivation and Affects Response to Gefitinib"

    Article Title: Estrogen Receptor ? Exerts Tumor Repressive Functions in Human Malignant Pleural Mesothelioma via EGFR Inactivation and Affects Response to Gefitinib

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0014110

    ERβ associates with EGFR and caveolin 1. A ) Co-immunoprecipitation experiments were performed on REN cells treated 1 and 5 minutes with 5 ng/ml of human recombinant EGF. ERβ and caveolin 1 were detected by Western blot in immunoprecipitations of membrane associated EGFR. B ) Co-immunoprecipitation experiments were performed on mock and ERβ over-expressing REN cells treated 5 minutes with 5 ng/ml of human recombinant EGF. ERβ and caveolin 1 were detected by Western blot in immunoprecipitations of membrane associated EGFR. C ) Confocal double fluorescent microscopy analysis of red-labeled ERβ with green-labeled EGFR or caveolin 1 in mock- (left panel) or ERβ-transfected (right panel) REN cells treated or not 5 minutes with 5 ng/ml of human recombinant EGF. D ) Confocal fluorescent microscopy analysis showing the localization of green-labeled EGFR and phalloidin-TRITC labeled actin filaments in mock and in ERβ and ERα transfected REN cells. Nuclei were counterstained with DAPI.
    Figure Legend Snippet: ERβ associates with EGFR and caveolin 1. A ) Co-immunoprecipitation experiments were performed on REN cells treated 1 and 5 minutes with 5 ng/ml of human recombinant EGF. ERβ and caveolin 1 were detected by Western blot in immunoprecipitations of membrane associated EGFR. B ) Co-immunoprecipitation experiments were performed on mock and ERβ over-expressing REN cells treated 5 minutes with 5 ng/ml of human recombinant EGF. ERβ and caveolin 1 were detected by Western blot in immunoprecipitations of membrane associated EGFR. C ) Confocal double fluorescent microscopy analysis of red-labeled ERβ with green-labeled EGFR or caveolin 1 in mock- (left panel) or ERβ-transfected (right panel) REN cells treated or not 5 minutes with 5 ng/ml of human recombinant EGF. D ) Confocal fluorescent microscopy analysis showing the localization of green-labeled EGFR and phalloidin-TRITC labeled actin filaments in mock and in ERβ and ERα transfected REN cells. Nuclei were counterstained with DAPI.

    Techniques Used: Immunoprecipitation, Recombinant, Western Blot, Expressing, Microscopy, Labeling, Transfection

    ERβ over-expression influences EGFR mediated signaling and internalization. A ) The graph show the growth curves of mock- and ERβ-transfected REN cell treated for 24 and 48 hours with 5 ng/ml of EGF in 2% FBS culture medium. At each time point, the cells were assayed for proliferation. Each value represents mean ± SD (n = 3). Adjacent to the graph is reported a representative Western blot analysis that documents ERβ expression. Tubulin staining indicates equal loading of the proteins. B ) Mock- and ERβ- transfected REN cells made quiescent for 2 hours were treated with 5 ng/ml of EGF for 5 minutes and detergent extracted. Levels of phosphorylated EGFR, ERK 1/2 MAP kinases and Akt were analyzed by immunoblotting. Membranes were also blotted with antibodies to EGFR, Erk1/2 and Akt to evaluate protein expression. Tubulin was blotted to show equal amount of loading. Western blot analysis with anti ERβ antibodies documents its expression in transfected cells. Representative of three separate experiments. C ) Evaluation of EGFR internalization was performed by Flow cytometry analysis on wild type and ERβ expressing REN cells treated 60 or 120 minutes with 10 ng/ml of human recombinant EGF. Histograms represent percentage of positive cells following incubation with anti-EGFR antibody indicated for each condition ± SD. Data are representative of three separate experiments. D ) Representative immunoprecipitation experiment of membrane associated EGFR performed on mock and ERβ over-expressing REN cells, treated 60 or 120 minutes with 10 ng/ml of human recombinant EGF. Membrane was blotted with anti-pY and anti-EGFR antibodies.
    Figure Legend Snippet: ERβ over-expression influences EGFR mediated signaling and internalization. A ) The graph show the growth curves of mock- and ERβ-transfected REN cell treated for 24 and 48 hours with 5 ng/ml of EGF in 2% FBS culture medium. At each time point, the cells were assayed for proliferation. Each value represents mean ± SD (n = 3). Adjacent to the graph is reported a representative Western blot analysis that documents ERβ expression. Tubulin staining indicates equal loading of the proteins. B ) Mock- and ERβ- transfected REN cells made quiescent for 2 hours were treated with 5 ng/ml of EGF for 5 minutes and detergent extracted. Levels of phosphorylated EGFR, ERK 1/2 MAP kinases and Akt were analyzed by immunoblotting. Membranes were also blotted with antibodies to EGFR, Erk1/2 and Akt to evaluate protein expression. Tubulin was blotted to show equal amount of loading. Western blot analysis with anti ERβ antibodies documents its expression in transfected cells. Representative of three separate experiments. C ) Evaluation of EGFR internalization was performed by Flow cytometry analysis on wild type and ERβ expressing REN cells treated 60 or 120 minutes with 10 ng/ml of human recombinant EGF. Histograms represent percentage of positive cells following incubation with anti-EGFR antibody indicated for each condition ± SD. Data are representative of three separate experiments. D ) Representative immunoprecipitation experiment of membrane associated EGFR performed on mock and ERβ over-expressing REN cells, treated 60 or 120 minutes with 10 ng/ml of human recombinant EGF. Membrane was blotted with anti-pY and anti-EGFR antibodies.

    Techniques Used: Over Expression, Transfection, Western Blot, Expressing, Staining, Flow Cytometry, Cytometry, Recombinant, Incubation, Immunoprecipitation

    12) Product Images from "Loss of Activating EGFR Mutant Gene Contributes to Acquired Resistance to EGFR Tyrosine Kinase Inhibitors in Lung Cancer Cells"

    Article Title: Loss of Activating EGFR Mutant Gene Contributes to Acquired Resistance to EGFR Tyrosine Kinase Inhibitors in Lung Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0041017

    The effect of erlotinib or knockdown of EGFR, HER2, or HER3 by their siRNAs on PC9/ER1 cells. A, Exponentially growing PC9 and PC9/ER1 cells were exposed to various doses of erlotinib for 5 h, and followed by western blot analysis. B, C, D, PC9 and PC9/ER1 cells were treated for 48 hr with 10 nM scramble (sc) siRNA, 2 nM or 10 nM EGFR siRNA, HER2 siRNA or HER3 siRNA respectively, and followed by Western blot analysis.
    Figure Legend Snippet: The effect of erlotinib or knockdown of EGFR, HER2, or HER3 by their siRNAs on PC9/ER1 cells. A, Exponentially growing PC9 and PC9/ER1 cells were exposed to various doses of erlotinib for 5 h, and followed by western blot analysis. B, C, D, PC9 and PC9/ER1 cells were treated for 48 hr with 10 nM scramble (sc) siRNA, 2 nM or 10 nM EGFR siRNA, HER2 siRNA or HER3 siRNA respectively, and followed by Western blot analysis.

    Techniques Used: Western Blot

    Our hypothetic model how drug resistance to erlotinib is acquired in lung cancer cells harboring activated mutant EGFR (mEGFR). Cell proliferation and survival of human lung cancer cells harboring activated mutant EGFR (PC9 and 11–18 cells) closely depend upon EGFR-driven PI3K/Akt pathway, and this proliferation/survival is highly susceptible to erlotinib and other EGFR TKIs. First, there is partial or complete loss of mEGFR gene allele in drug-resistant cell lines, and then gain of addiction to HER2/HER3 and PI3K/Akt signaling (PC9/ER1 cells). However, more definitive analysis on resistant cell lines of 11–18 is required because 11–18 resistant cell lines show only partial loss of mEGFR.
    Figure Legend Snippet: Our hypothetic model how drug resistance to erlotinib is acquired in lung cancer cells harboring activated mutant EGFR (mEGFR). Cell proliferation and survival of human lung cancer cells harboring activated mutant EGFR (PC9 and 11–18 cells) closely depend upon EGFR-driven PI3K/Akt pathway, and this proliferation/survival is highly susceptible to erlotinib and other EGFR TKIs. First, there is partial or complete loss of mEGFR gene allele in drug-resistant cell lines, and then gain of addiction to HER2/HER3 and PI3K/Akt signaling (PC9/ER1 cells). However, more definitive analysis on resistant cell lines of 11–18 is required because 11–18 resistant cell lines show only partial loss of mEGFR.

    Techniques Used: Mutagenesis

    Comparison of protein expression of EGFR family proteins and the down-stream molecules in erlotinib-resistant cell lines in the absence or presence of erlotinib. A, Western blot analysis showing the expression of pEGFR, EGFR, pHER2, HER2, pHER3, HER3, pc-Met, c-Met, PTEN, pAkt, Akt, pERK1/2, and ERK1/2 proteins, and α-tubulin as a loading control. B, Exponentially growing PC9 and PC9/ER1 cells were exposed to various doses of erlotinib for 5 hr, and followed by Western blot analysis. C, Exponentially growing 11–18, 11–18/ER1-7, and 11–18/ER2-1 cells were exposed to various doses of erlotinib for 5 hr, and followed by Western blot analysis.
    Figure Legend Snippet: Comparison of protein expression of EGFR family proteins and the down-stream molecules in erlotinib-resistant cell lines in the absence or presence of erlotinib. A, Western blot analysis showing the expression of pEGFR, EGFR, pHER2, HER2, pHER3, HER3, pc-Met, c-Met, PTEN, pAkt, Akt, pERK1/2, and ERK1/2 proteins, and α-tubulin as a loading control. B, Exponentially growing PC9 and PC9/ER1 cells were exposed to various doses of erlotinib for 5 hr, and followed by Western blot analysis. C, Exponentially growing 11–18, 11–18/ER1-7, and 11–18/ER2-1 cells were exposed to various doses of erlotinib for 5 hr, and followed by Western blot analysis.

    Techniques Used: Expressing, Western Blot

    13) Product Images from "Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells, et al. Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells"

    Article Title: Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells, et al. Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14730

    Expression level of EGFR and MET in generated variants of A375 and WM9 melanoma cell lines. A, Results of qRT‐PCR analysis of EGFR and MET expression are shown as the mean (relative expression compared to GAPDH) ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001. B, Western blotting analysis of EGFR and MET protein level in generated cell lines. Membranes were probed with antibodies directed against total EGFR and MET, and are representative for at least three independent experiments. GAPDH was used as the sample loading control
    Figure Legend Snippet: Expression level of EGFR and MET in generated variants of A375 and WM9 melanoma cell lines. A, Results of qRT‐PCR analysis of EGFR and MET expression are shown as the mean (relative expression compared to GAPDH) ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001. B, Western blotting analysis of EGFR and MET protein level in generated cell lines. Membranes were probed with antibodies directed against total EGFR and MET, and are representative for at least three independent experiments. GAPDH was used as the sample loading control

    Techniques Used: Expressing, Generated, Quantitative RT-PCR, Western Blot

    Proteolytic activity of melanoma cells with altered EGFR and MET expression. A, Representative pictures of proteolytic activity of A375 (MOCK, EGFR, shCTRL and shMET) and WM9 (shCTRL, shEGFR and shMET) cells (F‐actin visible in red) detected using FITC‐conjugated gelatin (green). Gelatin degradation indicated with white arrows is visualized as the dark areas on the fluorescently labelled gelatin background. Scale bar—10 µm. B, Quantification of digestion area calculated using ImageJ software from at least 40 cells from three independent experiments. Results are presented as the mean ± SD. C, MMP‐9 activity in concentrated conditioned media tested by gelatin zymography with (D) densitometric analysis. Results are expressed as the mean ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001
    Figure Legend Snippet: Proteolytic activity of melanoma cells with altered EGFR and MET expression. A, Representative pictures of proteolytic activity of A375 (MOCK, EGFR, shCTRL and shMET) and WM9 (shCTRL, shEGFR and shMET) cells (F‐actin visible in red) detected using FITC‐conjugated gelatin (green). Gelatin degradation indicated with white arrows is visualized as the dark areas on the fluorescently labelled gelatin background. Scale bar—10 µm. B, Quantification of digestion area calculated using ImageJ software from at least 40 cells from three independent experiments. Results are presented as the mean ± SD. C, MMP‐9 activity in concentrated conditioned media tested by gelatin zymography with (D) densitometric analysis. Results are expressed as the mean ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001

    Techniques Used: Activity Assay, Expressing, Software, Zymography

    Impact of EGFR and MET on invasion abilities and invadopodia formation in examined melanoma cell lines. A, The invasion assay performed on transwell filters coated with Matrigel for 24 h. Relative invasion factor was calculated versus control cells, where number of invading control cells is set as 100%. Results are expressed as the mean ± SD of three independent experiments. B, Representative pictures of A375 (MOCK, EGFR, shCTRL and shMET) and WM9 (shCTRL, shEGFR and shMET) cells seeded on Matrigel‐coated coverslips stained for F‐actin (red), cortactin (green) and cell nuclei (blue). Arrows indicate invadopodia. Scale bar—8 μm. C, Quantification of the average number of invadopodia in examined cells. Invadopodia formed by at least 40 cells from three independent experiments were counted, and results are presented as the mean ± SD. (**) P ≤ .01, (***) P ≤ .001
    Figure Legend Snippet: Impact of EGFR and MET on invasion abilities and invadopodia formation in examined melanoma cell lines. A, The invasion assay performed on transwell filters coated with Matrigel for 24 h. Relative invasion factor was calculated versus control cells, where number of invading control cells is set as 100%. Results are expressed as the mean ± SD of three independent experiments. B, Representative pictures of A375 (MOCK, EGFR, shCTRL and shMET) and WM9 (shCTRL, shEGFR and shMET) cells seeded on Matrigel‐coated coverslips stained for F‐actin (red), cortactin (green) and cell nuclei (blue). Arrows indicate invadopodia. Scale bar—8 μm. C, Quantification of the average number of invadopodia in examined cells. Invadopodia formed by at least 40 cells from three independent experiments were counted, and results are presented as the mean ± SD. (**) P ≤ .01, (***) P ≤ .001

    Techniques Used: Invasion Assay, Staining

    Migration abilities of melanoma cells with altered level of EGFR and MET. A, Cell trajectories and (B) migration distances of single A375 and WM9 cells analysed for 48 h using IncuCyte ® Live‐Cell Analysis System and ImageJ software. C, Representative images of wound closure, which was (D) quantified as per cent of area colonized by cells within 48 h (based on pictures analysed with an IncuCyte ® Scratch Wound Cell Migration Software Module). E, The migration assay executed on transwell filters for 24 h. Relative migration factor was calculated versus control cells, where number of migrating control cells is set as 100%. Results are expressed as the mean ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001
    Figure Legend Snippet: Migration abilities of melanoma cells with altered level of EGFR and MET. A, Cell trajectories and (B) migration distances of single A375 and WM9 cells analysed for 48 h using IncuCyte ® Live‐Cell Analysis System and ImageJ software. C, Representative images of wound closure, which was (D) quantified as per cent of area colonized by cells within 48 h (based on pictures analysed with an IncuCyte ® Scratch Wound Cell Migration Software Module). E, The migration assay executed on transwell filters for 24 h. Relative migration factor was calculated versus control cells, where number of migrating control cells is set as 100%. Results are expressed as the mean ± SD of three independent experiments. (**) P ≤ .01, (***) P ≤ .001

    Techniques Used: Migration, Software

    14) Product Images from "In Vitro and In Vivo Antitumor Activity of a Novel Semisynthetic Derivative of Cucurbitacin B"

    Article Title: In Vitro and In Vivo Antitumor Activity of a Novel Semisynthetic Derivative of Cucurbitacin B

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0117794

    Effects of DACE on TNFα-mediated activation of signaling pathways. (A) Effect of DACE on the phosphorylation status of AKT and ERK. (B) Effect of DACE on the phosphorylation status of AKT and ERK in A549 cells transiently transfected with 1μg of wild-type form of AKT or the empty pCMV5 vector. (C) Effect of DACE on the phosphorylation status of PI3K and its regulators PTEN and PDK1. In A, B, and C, the cells were exposed to 0.5μM and 1.0μM of DACE for 24h, stimulated or not for an additional 15 min with 30ng/mL TNFα and analyzed by Western blotting. (D) Effect of DACE on the phosphorylation level of EGFR, measured by phosphorylation of its specific Tyr 1068 site and downstream targets AKT and ERK. The A549 cells were transiently transfected with 1μg human EGFR or its comparable empty-vector control. The cells were exposed to 0.5μM and 1.0μM of DACE for 24h, stimulated or not with EGF (10ng/mL, 15min) and then analyzed by Western blotting. (E, F and G) Effect of DACE on the phosphorylation status of ERK in NIH3T3(k-RAS)- (E), NIH3T3(v-RAF)- transformed cells (F), and NIH3T3(wild-type) cells (G). The cells were simultaneously stimulated with TNFα (30ng/mL) and exposed or not to 1.0μM DACE for timepoints indicated and analyzed by Western blotting. Equal protein loading was confirmed by probing for tubulin or ERK2. The most representative results of three independent experiments are shown.
    Figure Legend Snippet: Effects of DACE on TNFα-mediated activation of signaling pathways. (A) Effect of DACE on the phosphorylation status of AKT and ERK. (B) Effect of DACE on the phosphorylation status of AKT and ERK in A549 cells transiently transfected with 1μg of wild-type form of AKT or the empty pCMV5 vector. (C) Effect of DACE on the phosphorylation status of PI3K and its regulators PTEN and PDK1. In A, B, and C, the cells were exposed to 0.5μM and 1.0μM of DACE for 24h, stimulated or not for an additional 15 min with 30ng/mL TNFα and analyzed by Western blotting. (D) Effect of DACE on the phosphorylation level of EGFR, measured by phosphorylation of its specific Tyr 1068 site and downstream targets AKT and ERK. The A549 cells were transiently transfected with 1μg human EGFR or its comparable empty-vector control. The cells were exposed to 0.5μM and 1.0μM of DACE for 24h, stimulated or not with EGF (10ng/mL, 15min) and then analyzed by Western blotting. (E, F and G) Effect of DACE on the phosphorylation status of ERK in NIH3T3(k-RAS)- (E), NIH3T3(v-RAF)- transformed cells (F), and NIH3T3(wild-type) cells (G). The cells were simultaneously stimulated with TNFα (30ng/mL) and exposed or not to 1.0μM DACE for timepoints indicated and analyzed by Western blotting. Equal protein loading was confirmed by probing for tubulin or ERK2. The most representative results of three independent experiments are shown.

    Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Western Blot, Transformation Assay

    15) Product Images from "Breast tumor kinase (Brk/PTK6) plays a role in the differentiation of primary keratinocytes"

    Article Title: Breast tumor kinase (Brk/PTK6) plays a role in the differentiation of primary keratinocytes

    Journal: Archives of Dermatological Research

    doi: 10.1007/s00403-010-1118-4

    Brk suppression induces chromatin fragmentation and suppression of EGFR. Brk levels were suppressed in normal human primary keratinocytes by RNAi. a 48 h following the second transfection with control (Control) or Brk-targeting siRNA (Brk siRNA), cells were harvested and assayed for chromatin fragmentation. As a positive control, primary keratinocytes were also cultured in methyl cellulose (MC) for 48 h prior to harvesting and assaying with the cell death ELISA. * P = 0.036. b Parallel wells were also lysed in hot SDS-PAGE lysis buffer and relative protein levels determined by western blotting
    Figure Legend Snippet: Brk suppression induces chromatin fragmentation and suppression of EGFR. Brk levels were suppressed in normal human primary keratinocytes by RNAi. a 48 h following the second transfection with control (Control) or Brk-targeting siRNA (Brk siRNA), cells were harvested and assayed for chromatin fragmentation. As a positive control, primary keratinocytes were also cultured in methyl cellulose (MC) for 48 h prior to harvesting and assaying with the cell death ELISA. * P = 0.036. b Parallel wells were also lysed in hot SDS-PAGE lysis buffer and relative protein levels determined by western blotting

    Techniques Used: Transfection, Positive Control, Cell Culture, Enzyme-linked Immunosorbent Assay, SDS Page, Lysis, Western Blot

    16) Product Images from "Inhibition of EGFR or IGF-1R signaling enhances radiation response in head and neck cancer models but concurrent inhibition has no added benefit"

    Article Title: Inhibition of EGFR or IGF-1R signaling enhances radiation response in head and neck cancer models but concurrent inhibition has no added benefit

    Journal: Cancer Medicine

    doi: 10.1002/cam4.345

    (A) Effect of IMC-A12 on IGF-1R and EGFR expression levels. Cells were exposed to IMC-A12and subjected to Western blot analysis. Numbers shown below protein bands are relative intensities with levels in untreated control cells as 1.0. Western blots shown are representative of two independent experiments. (B). Effect of cetuximab and IMC-A12 on radiosensitivity of HNSCC lines in culture. Cells were treated with cetuximab and/or IMC-A12 and exposed to radiation as described under Methods. Survival curves were constructed with normalized values for the cytotoxicity induced by cetuximab/IMC-A12. Data shown are means ± SE from three independent experiments. RT only: black solid line; cetuximab + RT: green dash line; IMC-A12 + RT: blue dash line; cetuximab + IMC-A12 + RT: Red solid line.
    Figure Legend Snippet: (A) Effect of IMC-A12 on IGF-1R and EGFR expression levels. Cells were exposed to IMC-A12and subjected to Western blot analysis. Numbers shown below protein bands are relative intensities with levels in untreated control cells as 1.0. Western blots shown are representative of two independent experiments. (B). Effect of cetuximab and IMC-A12 on radiosensitivity of HNSCC lines in culture. Cells were treated with cetuximab and/or IMC-A12 and exposed to radiation as described under Methods. Survival curves were constructed with normalized values for the cytotoxicity induced by cetuximab/IMC-A12. Data shown are means ± SE from three independent experiments. RT only: black solid line; cetuximab + RT: green dash line; IMC-A12 + RT: blue dash line; cetuximab + IMC-A12 + RT: Red solid line.

    Techniques Used: Expressing, Western Blot, Construct

    (A) Effect of cetuximab on EGFR and IGF-1R expression levels. Cells were exposed to cetuximab and subjected to Western blot analysis. Numbers shown below protein bands are relative intensities with levels in untreated control cells as 1.0. Western blots shown are representative of two independent experiments. (B) Effect of cetuximab on p-MAPK and p-Akt expression levels. Cells were exposed to cetuximab and subjected to Western blot analysis. Numbers shown below protein bands are relative intensities with levels in untreated control cells as 1.0. Western blots shown are representative of two independent experiments. (C) Effect of cetuximab, radiation, or both on dimerization of EGFR and IGF1R. Cells were exposed to either cetuximab and/or 4 Gy and collected 10 min after irradiation. Whole cell lysates were subjected to immunoprecipitation (IP) with IGF-1R antibody and immunoblotted (IB) with EGFR antibody. Shown are representative Western blots of two independent experiments. Cont: Untreated control; cetux: cetuximab. p-MAPK: phosphorylated form of MAPK; p-AKT: phosphorylated form of Akt.
    Figure Legend Snippet: (A) Effect of cetuximab on EGFR and IGF-1R expression levels. Cells were exposed to cetuximab and subjected to Western blot analysis. Numbers shown below protein bands are relative intensities with levels in untreated control cells as 1.0. Western blots shown are representative of two independent experiments. (B) Effect of cetuximab on p-MAPK and p-Akt expression levels. Cells were exposed to cetuximab and subjected to Western blot analysis. Numbers shown below protein bands are relative intensities with levels in untreated control cells as 1.0. Western blots shown are representative of two independent experiments. (C) Effect of cetuximab, radiation, or both on dimerization of EGFR and IGF1R. Cells were exposed to either cetuximab and/or 4 Gy and collected 10 min after irradiation. Whole cell lysates were subjected to immunoprecipitation (IP) with IGF-1R antibody and immunoblotted (IB) with EGFR antibody. Shown are representative Western blots of two independent experiments. Cont: Untreated control; cetux: cetuximab. p-MAPK: phosphorylated form of MAPK; p-AKT: phosphorylated form of Akt.

    Techniques Used: Expressing, Western Blot, Irradiation, Immunoprecipitation

    17) Product Images from "c-Src Associates with ErbB2 through an Interaction between Catalytic Domains and Confers Enhanced Transforming Potential ▿c-Src Associates with ErbB2 through an Interaction between Catalytic Domains and Confers Enhanced Transforming Potential ▿ †"

    Article Title: c-Src Associates with ErbB2 through an Interaction between Catalytic Domains and Confers Enhanced Transforming Potential ▿c-Src Associates with ErbB2 through an Interaction between Catalytic Domains and Confers Enhanced Transforming Potential ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01731-08

    EGFR YHAD specifically activates Stat3 in Rat1 cells. (A) Cells were serum deprived for 48 h and stimulated with EGF (10 ng/ml) for the indicated times. The lysates from EGFR- and EGFR YHAD -expressing Rat1 fibroblasts were subjected to Western blotting and probed for the specified proteins. (B) Rat1 fibroblasts were serum deprived for 48 h and stimulated with EGF for the indicated times. In the c-Src inhibition experiment, cells were pretreated with PP2 (10 μM) for 30 min prior to EGF stimulation. (C) RT-PCR analysis of EGFR- and EGFR YHAD -expressing Rat1 fibroblasts. Rat1 fibroblasts were serum deprived for 48 h and stimulated with EGF for the indicated times. RNA was extracted using Qiagen RNeasy following the manufacturer's instructions and subjected to RT-PCR as described in Materials and Methods. (D) EGFR-, EGFR TK -, and EGFR YHAD -expressing Rat1 fibroblasts were subjected to a focus transformation assay as described in Materials and Methods. The cells were cotransfected with DN-Stat3, as well as the indicated construct. The cells were provided with 10 ng/ml EGF daily. The error bars indicate standard deviations. EV, empty vector.
    Figure Legend Snippet: EGFR YHAD specifically activates Stat3 in Rat1 cells. (A) Cells were serum deprived for 48 h and stimulated with EGF (10 ng/ml) for the indicated times. The lysates from EGFR- and EGFR YHAD -expressing Rat1 fibroblasts were subjected to Western blotting and probed for the specified proteins. (B) Rat1 fibroblasts were serum deprived for 48 h and stimulated with EGF for the indicated times. In the c-Src inhibition experiment, cells were pretreated with PP2 (10 μM) for 30 min prior to EGF stimulation. (C) RT-PCR analysis of EGFR- and EGFR YHAD -expressing Rat1 fibroblasts. Rat1 fibroblasts were serum deprived for 48 h and stimulated with EGF for the indicated times. RNA was extracted using Qiagen RNeasy following the manufacturer's instructions and subjected to RT-PCR as described in Materials and Methods. (D) EGFR-, EGFR TK -, and EGFR YHAD -expressing Rat1 fibroblasts were subjected to a focus transformation assay as described in Materials and Methods. The cells were cotransfected with DN-Stat3, as well as the indicated construct. The cells were provided with 10 ng/ml EGF daily. The error bars indicate standard deviations. EV, empty vector.

    Techniques Used: Expressing, Western Blot, Inhibition, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Construct, Plasmid Preparation

    (A) EGFR somatic mutants identified in lung adenocarcinoma associate with c-Src. 293T cells were transfected with the indicated EGFR mutants, as well as wild-type c-Src. The lysates from these cells were subjected to coimmunoprecipitation (IP) experiments and probed for the indicated proteins. WB, Western blot. (B) 293T cells were transfected with EGFR TK or EGFR TK1 , as well as c-Src. The cells were serum deprived for 24 h and stimulated with EGF (10 ng/ml) for the indicated time (0, 1, or 2 h). To inhibit Src activity, the cells were pretreated for 30 min with 10 μM of PP2 before EGF stimulation. Phospho-EGFR corresponds to phospho-Y845. (C) Rat1 transformation assay. The numbers are averages of two independent assays. Medium with or without PP2 or SKI-1 supplemented with 10 ng/ml of EGF was changed every 3 days. SD, standard deviation.
    Figure Legend Snippet: (A) EGFR somatic mutants identified in lung adenocarcinoma associate with c-Src. 293T cells were transfected with the indicated EGFR mutants, as well as wild-type c-Src. The lysates from these cells were subjected to coimmunoprecipitation (IP) experiments and probed for the indicated proteins. WB, Western blot. (B) 293T cells were transfected with EGFR TK or EGFR TK1 , as well as c-Src. The cells were serum deprived for 24 h and stimulated with EGF (10 ng/ml) for the indicated time (0, 1, or 2 h). To inhibit Src activity, the cells were pretreated for 30 min with 10 μM of PP2 before EGF stimulation. Phospho-EGFR corresponds to phospho-Y845. (C) Rat1 transformation assay. The numbers are averages of two independent assays. Medium with or without PP2 or SKI-1 supplemented with 10 ng/ml of EGF was changed every 3 days. SD, standard deviation.

    Techniques Used: Transfection, Western Blot, Activity Assay, Transformation Assay, Standard Deviation

    18) Product Images from "Adhesion-Mediated Squamous Cell Carcinoma Survival through Ligand-Independent Activation of Epidermal Growth Factor Receptor"

    Article Title: Adhesion-Mediated Squamous Cell Carcinoma Survival through Ligand-Independent Activation of Epidermal Growth Factor Receptor

    Journal: The American Journal of Pathology

    doi:

    Cell-cell adhesion-induced EGFR activation suppresses anoikis. A: DNA fragmentation of HSC-3 cells. HSC-3 cells were plated as MCA culture in the absence or presence of 1 μg/ml AG1478 for 48 hours ( lanes 2 and 3 ). Suspended single cells (SC) alone ( lane 4 ) and monolayers (ML) cultured for 48 hours in the presence of 1 μmol/L AG1478 ( lane 5 ) were used as controls. The DNA laddering assay for intranucleosomal DNA cleavage was performed as described in Experimental Procedures. Lane 1 : Standard 100-bp DNA ladder. B: TUNEL analysis of AG1478-treated HSC-3 cells. HSC-3 cells were plated as ML, MCA, or SC culture for 48 hours before TUNEL analysis. AG1478 (1 μmol/L) was added to the culture as indicated. Values represent the apoptotic cell fractions (%) that stained positive with FITC-dUTP.
    Figure Legend Snippet: Cell-cell adhesion-induced EGFR activation suppresses anoikis. A: DNA fragmentation of HSC-3 cells. HSC-3 cells were plated as MCA culture in the absence or presence of 1 μg/ml AG1478 for 48 hours ( lanes 2 and 3 ). Suspended single cells (SC) alone ( lane 4 ) and monolayers (ML) cultured for 48 hours in the presence of 1 μmol/L AG1478 ( lane 5 ) were used as controls. The DNA laddering assay for intranucleosomal DNA cleavage was performed as described in Experimental Procedures. Lane 1 : Standard 100-bp DNA ladder. B: TUNEL analysis of AG1478-treated HSC-3 cells. HSC-3 cells were plated as ML, MCA, or SC culture for 48 hours before TUNEL analysis. AG1478 (1 μmol/L) was added to the culture as indicated. Values represent the apoptotic cell fractions (%) that stained positive with FITC-dUTP.

    Techniques Used: Activation Assay, Cell Culture, DNA Laddering, TUNEL Assay, Staining

    E-cadherin-mediated cell-cell adhesion induces EGFR activation. A: HSC-3 cells were cultured as MCAs for 0.5 hours ( a ), 3 hours ( b , c ), or 6 hours ( d , e ) and then transferred onto glass coverslips and immunolabeled for EGFR, E-cadherin, or activated EGFR (p-EGFR) as indicated. In C , which represents the early stage of cell aggregation, the top frame stained for E-cadherin is of a different cell pair than the others. Specimens were analyzed by confocal microscopy, and representative images are shown. B: Immunoblot analysis of activated EGFR during MCA formation. As controls, HSC-3 cells were cultured in suspension as single cells (SCs) for 24 hours or treated with 1 μmol/L tyrphostin AG1478 for 24 hours in MCA culture. Relative protein densities of phosphorylated EGFR were determined; EGFR kinase activity is expressed as fold induction relative to the HSC-3 MCA at 0 hours. Data reported here are representative of four independent experiments. C: Effect of anti-E-cadherin mAb on HSC-3 MCA formation. HSC-3 cells were plated as MCA culture in the absence or presence of 50 μg/ml anti-E-cadherin mAb HECD-1 for 24 hours. D: HSC-3 cells were plated as MCA culture in the presence of 50 μg/ml anti-E-cadherin mAb HECD-1 or mouse IgG for 24 hours. Equivalent protein was immunoblotted with mAbs to phospho-EGFR and total EGFR.
    Figure Legend Snippet: E-cadherin-mediated cell-cell adhesion induces EGFR activation. A: HSC-3 cells were cultured as MCAs for 0.5 hours ( a ), 3 hours ( b , c ), or 6 hours ( d , e ) and then transferred onto glass coverslips and immunolabeled for EGFR, E-cadherin, or activated EGFR (p-EGFR) as indicated. In C , which represents the early stage of cell aggregation, the top frame stained for E-cadherin is of a different cell pair than the others. Specimens were analyzed by confocal microscopy, and representative images are shown. B: Immunoblot analysis of activated EGFR during MCA formation. As controls, HSC-3 cells were cultured in suspension as single cells (SCs) for 24 hours or treated with 1 μmol/L tyrphostin AG1478 for 24 hours in MCA culture. Relative protein densities of phosphorylated EGFR were determined; EGFR kinase activity is expressed as fold induction relative to the HSC-3 MCA at 0 hours. Data reported here are representative of four independent experiments. C: Effect of anti-E-cadherin mAb on HSC-3 MCA formation. HSC-3 cells were plated as MCA culture in the absence or presence of 50 μg/ml anti-E-cadherin mAb HECD-1 for 24 hours. D: HSC-3 cells were plated as MCA culture in the presence of 50 μg/ml anti-E-cadherin mAb HECD-1 or mouse IgG for 24 hours. Equivalent protein was immunoblotted with mAbs to phospho-EGFR and total EGFR.

    Techniques Used: Activation Assay, Cell Culture, Immunolabeling, Staining, Confocal Microscopy, Activity Assay

    Assembly and co-localization of the EGFR/E-cadherin complex is independent of EGFR kinase activity. A: HSC-3 cells were cultured as MCAs for 6 hours in the absence or presence of AG1478 (1 μmol/L) and then transferred onto glass coverslips and immunolabeled for EGFR (green) and E-cadherin or activated EGFR (p-EGFR) (red). Specimens were analyzed by confocal microscopy, and representative images are shown. B: HSC-3 cells were plated as MCA for 12 hours in the absence or presence of AG1478 (1 μmol/L). E-cadherin was immunoprecipitated (IP) from total cell lysates with HECD-1 mAb. The presence of activated EGFR (p-EGFR) or total EGFR in the immunoprecipitate was detected with specific anti-p-EGFR or anti-EGFR mAb.
    Figure Legend Snippet: Assembly and co-localization of the EGFR/E-cadherin complex is independent of EGFR kinase activity. A: HSC-3 cells were cultured as MCAs for 6 hours in the absence or presence of AG1478 (1 μmol/L) and then transferred onto glass coverslips and immunolabeled for EGFR (green) and E-cadherin or activated EGFR (p-EGFR) (red). Specimens were analyzed by confocal microscopy, and representative images are shown. B: HSC-3 cells were plated as MCA for 12 hours in the absence or presence of AG1478 (1 μmol/L). E-cadherin was immunoprecipitated (IP) from total cell lysates with HECD-1 mAb. The presence of activated EGFR (p-EGFR) or total EGFR in the immunoprecipitate was detected with specific anti-p-EGFR or anti-EGFR mAb.

    Techniques Used: Activity Assay, Cell Culture, Immunolabeling, Confocal Microscopy, Immunoprecipitation

    19) Product Images from "The scaffolding protein NHERF1 regulates the stability and activity of the tyrosine kinase HER2"

    Article Title: The scaffolding protein NHERF1 regulates the stability and activity of the tyrosine kinase HER2

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.770883

    A , HER2, NHERF1, and PMCA2 mRNA levels in MCF10A and SKBR3 cells as assessed by QPCR. B , confocal images of immunofluorescence co-staining of SKBR3 cells for NHERF1 ( green ) with PMCA2, HER2, or actin ( red ) as indicated. The images on the right show merged staining. The top and right panels of each image demonstrate optical sections in different orientations. Arrows point to apical membrane protrusions. Scale bars = 10 μm. C , confocal images of co-immunofluorescence for HER2 and actin (phalloidin) or NHERF1 in SKBR3 cells in serum-free medium ( SFM , top row ) or after treatment with EGF ( center row ) or NRG1 ( bottom row ). The top and right panels of each image represent optical sections through the cells in two different orientations. The left three columns show staining for HER2 ( red ), phalloidin ( green ), and merged staining. The right three columns show staining for HER2 ( green ), NHERF1 ( red ), and merged staining. Arrows point to the more prominent apical membrane protrusions that are actin-rich and contain HER2 and NHERF1 that form after treatment with growth factors. D , confocal images of co-immunofluorescence for NHERF1 ( green ) and EGFR in SKBR3 cells in growth medium ( top row ), in serum-free medium ( center row ), and following acute treatment with EGF. E , confocal images of co-immunofluorescence for NHERF1 ( green ) and HER3 ( red ) in SKBR3 cells in growth medium ( top row ), in serum-free medium ( center row ), and following acute treatment with NRG1. The top and right panels of each image depict optical sections through the cells in two different orientations. Scale Bars = 10 μm. F , confocal images of immunofluorescence for HER2 ( aqua ), PMCA2 ( green ), or NHERF1 ( red ) in MCF10A cells constitutively overexpressing HER2 and transfected with GFP-labeled, WT PMCA2, and FLAG-tagged NHERF1. The bottom right image shows merged images for all three stains. The top and right panels of each image demonstrate optical sections in different orientations. Boxed areas are magnified at the right. G , co-immunoprecipitation experiments in CHO or MCF10A cells transiently transfected with GFP-tagged PMCA2 and His-tagged NHERF1. All cells were transfected with either WT GFP-PMCA2 ( lanes 1 and 3 ) or Δ6-mutant GFP-PMCA2 lacking the C-terminal six amino acids that include the PDZ interaction motif ( lanes 2 and 4 ). Lanes 1 and 2 represent cells without NHERF1, whereas lanes 3 and 4 represent cells transfected with his-NHERF1. IB , immunoblot. H , co-immunoprecipitation of HER2 and FLAG-tagged NHERF1 in MCF10A cells with or without treatment with EGF. IP of FLAG-tagged NHERF1 from MCF10A cells pulled down HER2 only after acute treatment with EGF. The left panel represents the mean ± S.E. of the relative increase in immunoprecipitated HER2 after EGF in four different experiments. **, p
    Figure Legend Snippet: A , HER2, NHERF1, and PMCA2 mRNA levels in MCF10A and SKBR3 cells as assessed by QPCR. B , confocal images of immunofluorescence co-staining of SKBR3 cells for NHERF1 ( green ) with PMCA2, HER2, or actin ( red ) as indicated. The images on the right show merged staining. The top and right panels of each image demonstrate optical sections in different orientations. Arrows point to apical membrane protrusions. Scale bars = 10 μm. C , confocal images of co-immunofluorescence for HER2 and actin (phalloidin) or NHERF1 in SKBR3 cells in serum-free medium ( SFM , top row ) or after treatment with EGF ( center row ) or NRG1 ( bottom row ). The top and right panels of each image represent optical sections through the cells in two different orientations. The left three columns show staining for HER2 ( red ), phalloidin ( green ), and merged staining. The right three columns show staining for HER2 ( green ), NHERF1 ( red ), and merged staining. Arrows point to the more prominent apical membrane protrusions that are actin-rich and contain HER2 and NHERF1 that form after treatment with growth factors. D , confocal images of co-immunofluorescence for NHERF1 ( green ) and EGFR in SKBR3 cells in growth medium ( top row ), in serum-free medium ( center row ), and following acute treatment with EGF. E , confocal images of co-immunofluorescence for NHERF1 ( green ) and HER3 ( red ) in SKBR3 cells in growth medium ( top row ), in serum-free medium ( center row ), and following acute treatment with NRG1. The top and right panels of each image depict optical sections through the cells in two different orientations. Scale Bars = 10 μm. F , confocal images of immunofluorescence for HER2 ( aqua ), PMCA2 ( green ), or NHERF1 ( red ) in MCF10A cells constitutively overexpressing HER2 and transfected with GFP-labeled, WT PMCA2, and FLAG-tagged NHERF1. The bottom right image shows merged images for all three stains. The top and right panels of each image demonstrate optical sections in different orientations. Boxed areas are magnified at the right. G , co-immunoprecipitation experiments in CHO or MCF10A cells transiently transfected with GFP-tagged PMCA2 and His-tagged NHERF1. All cells were transfected with either WT GFP-PMCA2 ( lanes 1 and 3 ) or Δ6-mutant GFP-PMCA2 lacking the C-terminal six amino acids that include the PDZ interaction motif ( lanes 2 and 4 ). Lanes 1 and 2 represent cells without NHERF1, whereas lanes 3 and 4 represent cells transfected with his-NHERF1. IB , immunoblot. H , co-immunoprecipitation of HER2 and FLAG-tagged NHERF1 in MCF10A cells with or without treatment with EGF. IP of FLAG-tagged NHERF1 from MCF10A cells pulled down HER2 only after acute treatment with EGF. The left panel represents the mean ± S.E. of the relative increase in immunoprecipitated HER2 after EGF in four different experiments. **, p

    Techniques Used: Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Transfection, Labeling, Immunoprecipitation, Mutagenesis

    Model for the interaction of NHERF1 with PMCA2 and HER2 in breast cancer cells. a , at baseline, NHERF1 helps maintain PMCA2, HSP90, and HER2 in small, lipid raft-rich protrusions from the membrane. b , acute activation of HER2 leads to enlargement of the membrane protrusions with recruitment of NHERF1 to support interactions between PMCA2, HSP90, and HER2 that allow HER2 to remain within the protrusions, whereas EGFR and HER3 are internalized into the cell. c , in the absence of NHERF1, there is a reduction in PMCA2 expression and the lipid raft content of the membranes as well as an effacement of the membrane protrusions. In addition, upon stimulation, HER2 no longer remains at the cell surface but is internalized with EGFR and HER3.
    Figure Legend Snippet: Model for the interaction of NHERF1 with PMCA2 and HER2 in breast cancer cells. a , at baseline, NHERF1 helps maintain PMCA2, HSP90, and HER2 in small, lipid raft-rich protrusions from the membrane. b , acute activation of HER2 leads to enlargement of the membrane protrusions with recruitment of NHERF1 to support interactions between PMCA2, HSP90, and HER2 that allow HER2 to remain within the protrusions, whereas EGFR and HER3 are internalized into the cell. c , in the absence of NHERF1, there is a reduction in PMCA2 expression and the lipid raft content of the membranes as well as an effacement of the membrane protrusions. In addition, upon stimulation, HER2 no longer remains at the cell surface but is internalized with EGFR and HER3.

    Techniques Used: Activation Assay, Expressing

    A , immunofluorescence for HER2 ( green ) and EGFR ( red ) in control ( top row ) and NHERF1KD ( bottom row ) SKBR3 cells after treatment with EGF. B , immunofluorescence for HER2 ( green ) and HER3 ( red ) in control ( top row ) and NHERF1KD ( bottom row ) cells after treatment with NRG1. C , immunofluorescence for HER2 ( green ) and rab5 ( red ) in control ( top row ) and NHERF1KD cells ( bottom row ) after treatment with EGF. D , immunofluorescence for HER2 ( green ) and cbl ( red ) in control ( top row ) and NHERF1KD ( bottom row ) cells after treatment with EGF. E , immunofluorescence for HER2 ( green ) and polyubiquitin (FK2, red ) in control ( top row ) and NHERF1KD ( bottom row ) cells after treatment with EGF. F , immunofluorescence for pHER2 ( green ) and polyubiquitin (FK2, red ) in control ( top row ) and NHERF1KD ( bottom row ) after treatment with EGF. In A–F , the third panel in each row shows merged staining with DAPI ( blue ), and the fourth panel in each row represents a magnified view of the boxed region of the third panels. G , immunofluorescence for polyubiquitin (FK2, green ) and EGFR ( red ) after acute treatment of control or NHERF1KD cells with EGF. The images on the right show merged staining for both antibodies and DAPI ( blue ) to mark nuclei. H , immunofluorescence for polyubiquitin (FK2, green ) and HER3 ( red ) after acute treatment of control or NHERF1KD cells with NRG1. The images on the right show merged staining for both antibodies and DAPI ( blue ) to mark nuclei. Scale bars = 10 μm. I , co-IP for polyubiquitin complexes and HER2. IP for ubiquitinated proteins using FK2 antibody pulled down more HER2 from NHERF1KD cells than from control cells with or without treatment with EGF or NRG1. J , immunofluorescence for HER2 ( green ) and HSP90 ( red ) in control ( first and third rows ) and NHERF1KD ( second and fourth rows ) cells without ( first and second rows ) or with ( third and fourth rows ) treatment with EGF. The arrow points to internalized HER2, dissociated from HSP90, after EGF treatment of NHERF1KD cells. Scale bars = 10 μm. K , co-immunoprecipitation of HER2 and HSP90 from control and NHERF1KD cells. Less HER2 is pulled down with HSP90 in NHERF1 KD cells. IB , immunoblot.
    Figure Legend Snippet: A , immunofluorescence for HER2 ( green ) and EGFR ( red ) in control ( top row ) and NHERF1KD ( bottom row ) SKBR3 cells after treatment with EGF. B , immunofluorescence for HER2 ( green ) and HER3 ( red ) in control ( top row ) and NHERF1KD ( bottom row ) cells after treatment with NRG1. C , immunofluorescence for HER2 ( green ) and rab5 ( red ) in control ( top row ) and NHERF1KD cells ( bottom row ) after treatment with EGF. D , immunofluorescence for HER2 ( green ) and cbl ( red ) in control ( top row ) and NHERF1KD ( bottom row ) cells after treatment with EGF. E , immunofluorescence for HER2 ( green ) and polyubiquitin (FK2, red ) in control ( top row ) and NHERF1KD ( bottom row ) cells after treatment with EGF. F , immunofluorescence for pHER2 ( green ) and polyubiquitin (FK2, red ) in control ( top row ) and NHERF1KD ( bottom row ) after treatment with EGF. In A–F , the third panel in each row shows merged staining with DAPI ( blue ), and the fourth panel in each row represents a magnified view of the boxed region of the third panels. G , immunofluorescence for polyubiquitin (FK2, green ) and EGFR ( red ) after acute treatment of control or NHERF1KD cells with EGF. The images on the right show merged staining for both antibodies and DAPI ( blue ) to mark nuclei. H , immunofluorescence for polyubiquitin (FK2, green ) and HER3 ( red ) after acute treatment of control or NHERF1KD cells with NRG1. The images on the right show merged staining for both antibodies and DAPI ( blue ) to mark nuclei. Scale bars = 10 μm. I , co-IP for polyubiquitin complexes and HER2. IP for ubiquitinated proteins using FK2 antibody pulled down more HER2 from NHERF1KD cells than from control cells with or without treatment with EGF or NRG1. J , immunofluorescence for HER2 ( green ) and HSP90 ( red ) in control ( first and third rows ) and NHERF1KD ( second and fourth rows ) cells without ( first and second rows ) or with ( third and fourth rows ) treatment with EGF. The arrow points to internalized HER2, dissociated from HSP90, after EGF treatment of NHERF1KD cells. Scale bars = 10 μm. K , co-immunoprecipitation of HER2 and HSP90 from control and NHERF1KD cells. Less HER2 is pulled down with HSP90 in NHERF1 KD cells. IB , immunoblot.

    Techniques Used: Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Immunoprecipitation

    20) Product Images from "In Vitro and In Vivo Antitumor Activity of a Novel Semisynthetic Derivative of Cucurbitacin B"

    Article Title: In Vitro and In Vivo Antitumor Activity of a Novel Semisynthetic Derivative of Cucurbitacin B

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0117794

    Effects of DACE on TNFα-mediated activation of signaling pathways. (A) Effect of DACE on the phosphorylation status of AKT and ERK. (B) Effect of DACE on the phosphorylation status of AKT and ERK in A549 cells transiently transfected with 1μg of wild-type form of AKT or the empty pCMV5 vector. (C) Effect of DACE on the phosphorylation status of PI3K and its regulators PTEN and PDK1. In A, B, and C, the cells were exposed to 0.5μM and 1.0μM of DACE for 24h, stimulated or not for an additional 15 min with 30ng/mL TNFα and analyzed by Western blotting. (D) Effect of DACE on the phosphorylation level of EGFR, measured by phosphorylation of its specific Tyr 1068 site and downstream targets AKT and ERK. The A549 cells were transiently transfected with 1μg human EGFR or its comparable empty-vector control. The cells were exposed to 0.5μM and 1.0μM of DACE for 24h, stimulated or not with EGF (10ng/mL, 15min) and then analyzed by Western blotting. (E, F and G) Effect of DACE on the phosphorylation status of ERK in NIH3T3(k-RAS)- (E), NIH3T3(v-RAF)- transformed cells (F), and NIH3T3(wild-type) cells (G). The cells were simultaneously stimulated with TNFα (30ng/mL) and exposed or not to 1.0μM DACE for timepoints indicated and analyzed by Western blotting. Equal protein loading was confirmed by probing for tubulin or ERK2. The most representative results of three independent experiments are shown.
    Figure Legend Snippet: Effects of DACE on TNFα-mediated activation of signaling pathways. (A) Effect of DACE on the phosphorylation status of AKT and ERK. (B) Effect of DACE on the phosphorylation status of AKT and ERK in A549 cells transiently transfected with 1μg of wild-type form of AKT or the empty pCMV5 vector. (C) Effect of DACE on the phosphorylation status of PI3K and its regulators PTEN and PDK1. In A, B, and C, the cells were exposed to 0.5μM and 1.0μM of DACE for 24h, stimulated or not for an additional 15 min with 30ng/mL TNFα and analyzed by Western blotting. (D) Effect of DACE on the phosphorylation level of EGFR, measured by phosphorylation of its specific Tyr 1068 site and downstream targets AKT and ERK. The A549 cells were transiently transfected with 1μg human EGFR or its comparable empty-vector control. The cells were exposed to 0.5μM and 1.0μM of DACE for 24h, stimulated or not with EGF (10ng/mL, 15min) and then analyzed by Western blotting. (E, F and G) Effect of DACE on the phosphorylation status of ERK in NIH3T3(k-RAS)- (E), NIH3T3(v-RAF)- transformed cells (F), and NIH3T3(wild-type) cells (G). The cells were simultaneously stimulated with TNFα (30ng/mL) and exposed or not to 1.0μM DACE for timepoints indicated and analyzed by Western blotting. Equal protein loading was confirmed by probing for tubulin or ERK2. The most representative results of three independent experiments are shown.

    Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Western Blot, Transformation Assay

    21) Product Images from "Alternate Paths from Epidermal Growth Factor Receptor to Akt in Malignant Versus Nontransformed Lung Epithelial Cells"

    Article Title: Alternate Paths from Epidermal Growth Factor Receptor to Akt in Malignant Versus Nontransformed Lung Epithelial Cells

    Journal: American Journal of Respiratory Cell and Molecular Biology

    doi: 10.1165/rcmb.2005-0049OC

    Complexes containing Shp2 with Gab1 and p85 in C10 and E10 cells. ( A and B ) C10/A5 and E10/E9 paired nontransformed/malignant cell lines were serum starved and treated with TGF-α with or without EGFR inhibitor PD153035. Lysates were immunoprecipitated
    Figure Legend Snippet: Complexes containing Shp2 with Gab1 and p85 in C10 and E10 cells. ( A and B ) C10/A5 and E10/E9 paired nontransformed/malignant cell lines were serum starved and treated with TGF-α with or without EGFR inhibitor PD153035. Lysates were immunoprecipitated

    Techniques Used: Immunoprecipitation

    22) Product Images from "Expression and Pharmacological Inhibition of TrkB and EGFR in Glioblastoma"

    Article Title: Expression and Pharmacological Inhibition of TrkB and EGFR in Glioblastoma

    Journal: bioRxiv

    doi: 10.1101/2020.02.03.932608

    Inhibition of TrkB and EGFR alone or in combination reduces human GBM cell viability. Time course analysis of cell viability, by trypan blue cell counting, were performed after 24, 48 and 72 h of exposure to ANA-12 or AG 1478 exposure in A172 (A, B) and U87MG (C, D) cells. Dose-response curves were evaluated by trypan blue cell counting after treatment with increasing concentrations of ANA-12 (1-50μM) or AG 1478 (1-30 μM) for 48 h in A172 cells (E, F) and 24 h in U87MG cells (G, H). The drug vehicles (DMSO or EtOH) served as controls. Dose-response curves after combined treatment with ANA-12 and AG 1478 were evaluated after 48 h of drug exposure in A172 cells (I) and 24 h in U87MG cells (J). Data are expressed by mean ± SEM and represent three independent experiments * p
    Figure Legend Snippet: Inhibition of TrkB and EGFR alone or in combination reduces human GBM cell viability. Time course analysis of cell viability, by trypan blue cell counting, were performed after 24, 48 and 72 h of exposure to ANA-12 or AG 1478 exposure in A172 (A, B) and U87MG (C, D) cells. Dose-response curves were evaluated by trypan blue cell counting after treatment with increasing concentrations of ANA-12 (1-50μM) or AG 1478 (1-30 μM) for 48 h in A172 cells (E, F) and 24 h in U87MG cells (G, H). The drug vehicles (DMSO or EtOH) served as controls. Dose-response curves after combined treatment with ANA-12 and AG 1478 were evaluated after 48 h of drug exposure in A172 cells (I) and 24 h in U87MG cells (J). Data are expressed by mean ± SEM and represent three independent experiments * p

    Techniques Used: Inhibition, Cell Counting

    Synergistic effect after combined inhibition of TrkB and EGFR in A172 GBM cells. A172 and U87MG cell lines were treated with varying concentrations of ANA-12 and AG 1478 alone or in combination. The IC50-values were calculated from the dose-response curves after different exposure times (48 h for A172 and 24 h for U87MG cells) and expressed with their respective 95% confidence intervals and summarized in the table (A). The combination index (CI) was determined by the method of Chou-Talalay and data are presented as mean ± SEM (B).
    Figure Legend Snippet: Synergistic effect after combined inhibition of TrkB and EGFR in A172 GBM cells. A172 and U87MG cell lines were treated with varying concentrations of ANA-12 and AG 1478 alone or in combination. The IC50-values were calculated from the dose-response curves after different exposure times (48 h for A172 and 24 h for U87MG cells) and expressed with their respective 95% confidence intervals and summarized in the table (A). The combination index (CI) was determined by the method of Chou-Talalay and data are presented as mean ± SEM (B).

    Techniques Used: Inhibition

    Inhibition of TrkB and EGFR in an intracranial GBM mouse model. (A) A total of 40,000 U87MG cells were intracranially (i.c.) injected into nude mice. Drug treatments started on the seventh day after cell implantation. The animals were randomly divided in 4 groups ( n= 5 per group) to receive intraperitoneal (i.p.) injections for 21 days, and were treated by a blinded investigator with ANA-12 (1 mg/kg daily plus vehicle every 3 days), AG1478 (10 mg/kg every three days plus vehicle daily), ANA-12 (1 mg/kg daily) plus AG 1478 (10 mg/kg/every three days) and vehicle (DMSO) daily. After 21 days of treatment, the animals received an i.p. injection of 5-ALA (50 mg/kg) and after 1 h mice were euthanized by cervical dislocation and the brains were removed to be analyzed (B) Representative IVIS images of harvested brains acquired at day 29 after transplantation. (C) Corresponding data of 5-ALA (PPIX) radiant efficiency from the whole brain. (D) After performing images of the intact brain, a brain matrix was used to cut sequential 1-mm slices through the region containing the tumor. Slices were imaged using IVIS and fluorescence images were collected. Data are expressed as mean ± SEM. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post-hoc tests. No statistical differences were observed between experimental groups.
    Figure Legend Snippet: Inhibition of TrkB and EGFR in an intracranial GBM mouse model. (A) A total of 40,000 U87MG cells were intracranially (i.c.) injected into nude mice. Drug treatments started on the seventh day after cell implantation. The animals were randomly divided in 4 groups ( n= 5 per group) to receive intraperitoneal (i.p.) injections for 21 days, and were treated by a blinded investigator with ANA-12 (1 mg/kg daily plus vehicle every 3 days), AG1478 (10 mg/kg every three days plus vehicle daily), ANA-12 (1 mg/kg daily) plus AG 1478 (10 mg/kg/every three days) and vehicle (DMSO) daily. After 21 days of treatment, the animals received an i.p. injection of 5-ALA (50 mg/kg) and after 1 h mice were euthanized by cervical dislocation and the brains were removed to be analyzed (B) Representative IVIS images of harvested brains acquired at day 29 after transplantation. (C) Corresponding data of 5-ALA (PPIX) radiant efficiency from the whole brain. (D) After performing images of the intact brain, a brain matrix was used to cut sequential 1-mm slices through the region containing the tumor. Slices were imaged using IVIS and fluorescence images were collected. Data are expressed as mean ± SEM. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post-hoc tests. No statistical differences were observed between experimental groups.

    Techniques Used: Inhibition, Injection, Mouse Assay, Transplantation Assay, Fluorescence

    Inhibition of TrkB and EGFR alone or in combination in a subcutaneous GBM xenograft mouse model. U87MG cells were pretreated in vitro for 24 h with ANA-12 (13.85 μM), AG-1478 (13.26 μM) or ANA-12 plus AG-1478, and the viable cells were injected into the flanks of nude mice (6-7 mice per group) as shown in the schematic drawing (A). Caliper measurements were used to determine the displayed subcutaneous tumor volume. Mice were euthanized and tumors were excised when volume reached approximately 800-1,000 mm 3 (B) Tumors were measured every 2 days and volumes were calculated as described in materials and methods section. Tumor growth is represented by tumor volume (mm 3 ) at the indicated days; Control ( n = 7), ANA-12 ( n= 8), AG 1478 ( n= 8) and ANA-12 plus AG 1478 ( n= 8) (C) Tumor growth curve is shown on selected time points of 15, 31 and 45 days to highlight statistical differences (D). Tumor volumes (mm 3 ) at the time of tissue harvest. (E) Tumor weight (mg) at the time of tissue harvest are shown in (F) Kaplan-Meier curves presenting percent of mice surviving following tumor implantation. Data are expressed as mean ± SEM (* p
    Figure Legend Snippet: Inhibition of TrkB and EGFR alone or in combination in a subcutaneous GBM xenograft mouse model. U87MG cells were pretreated in vitro for 24 h with ANA-12 (13.85 μM), AG-1478 (13.26 μM) or ANA-12 plus AG-1478, and the viable cells were injected into the flanks of nude mice (6-7 mice per group) as shown in the schematic drawing (A). Caliper measurements were used to determine the displayed subcutaneous tumor volume. Mice were euthanized and tumors were excised when volume reached approximately 800-1,000 mm 3 (B) Tumors were measured every 2 days and volumes were calculated as described in materials and methods section. Tumor growth is represented by tumor volume (mm 3 ) at the indicated days; Control ( n = 7), ANA-12 ( n= 8), AG 1478 ( n= 8) and ANA-12 plus AG 1478 ( n= 8) (C) Tumor growth curve is shown on selected time points of 15, 31 and 45 days to highlight statistical differences (D). Tumor volumes (mm 3 ) at the time of tissue harvest. (E) Tumor weight (mg) at the time of tissue harvest are shown in (F) Kaplan-Meier curves presenting percent of mice surviving following tumor implantation. Data are expressed as mean ± SEM (* p

    Techniques Used: Inhibition, In Vitro, Injection, Mouse Assay, Tumor Implantation

    23) Product Images from "89Zr-Onartuzumab PET imaging of c-MET receptor dynamics"

    Article Title: 89Zr-Onartuzumab PET imaging of c-MET receptor dynamics

    Journal: European Journal of Nuclear Medicine and Molecular Imaging

    doi: 10.1007/s00259-017-3672-x

    a In vitro flow cytometric analysis of EGFR and c-MET membrane expression in HCC827ErlRes cells normalized to expression in parental cell line HCC827. b In vitro MTT proliferation assay in HCC827 and HCC827ErlRes cells with exposure to increasing concentrations of erlotinib for 4 days. c In vitro flow cytometric analysis of EGFR and c-MET membrane expression in HCC827 and HCC827ErlRes cells after 24 h treatment with 25, 50 and 100 nM NVP-AUY-922 normalized to untreated controls. d In vitro MTT proliferation assay in HCC827 and HCC827ErlRes cells with exposure to increasing concentrations of NVP-AUY-922 for 4 days
    Figure Legend Snippet: a In vitro flow cytometric analysis of EGFR and c-MET membrane expression in HCC827ErlRes cells normalized to expression in parental cell line HCC827. b In vitro MTT proliferation assay in HCC827 and HCC827ErlRes cells with exposure to increasing concentrations of erlotinib for 4 days. c In vitro flow cytometric analysis of EGFR and c-MET membrane expression in HCC827 and HCC827ErlRes cells after 24 h treatment with 25, 50 and 100 nM NVP-AUY-922 normalized to untreated controls. d In vitro MTT proliferation assay in HCC827 and HCC827ErlRes cells with exposure to increasing concentrations of NVP-AUY-922 for 4 days

    Techniques Used: In Vitro, Flow Cytometry, Expressing, MTT Assay, Proliferation Assay

    24) Product Images from "Constitutive and ligand-induced EGFR signaling triggers distinct and mutually exclusive downstream signaling networks"

    Article Title: Constitutive and ligand-induced EGFR signaling triggers distinct and mutually exclusive downstream signaling networks

    Journal: Nature communications

    doi: 10.1038/ncomms6811

    EGFR forms a signaling complex with IRF3 and TBK1 (a) EGFR co-immunoprecipitates with IRF3 in U251EGFR cells in the absence of EGF. When EGF is added there is a rapid loss of the EGFR-IRF3 association. (b) Similarly, the EGFR coimmunoprecipitates with TBK1 in U251EGFR cells. When EGF is added there is a rapid loss of the EGFR-TBK1 association. (c) TBK1 is associated with IRF3 in U251EGFR cells in the absence of EGF. When EGF is added to cells, there is a rapid decrease in TBK-IRF3 association. The TBK1-IRF3 association is also disrupted when Erlotinib is used suggesting that it is EGFR driven. C refers to control vehicle (PBS). (d) EGFR co-immunoprecipitates with IRF3 in MDAMB468 cells in the absence of EGF. When EGF is added there is a rapid loss of the EGFR-IRF3 association. (e) Similarly, the EGFR coimmunoprecipitates with TBK1 in MDAMB468 cells. When EGF is added there is a rapid loss of the EGFR-TBK1 association. (f) TBK1 is associated with IRF3 in MDAMB468 cells in the absence of EGF. When EGF is added to cells, there is a rapid decrease in TBK-IRF3 association. The TBK1-IRF3 association is also disrupted when Erlotinib is used suggesting that it is EGFR driven. (g) In contradistinction to IRF3 and TBK1, Shc becomes associated with the EGFR only when EGF is added to U251EGFR cells. (h) About 10% of IRF3 is associated with the EGFR. (i) About 5% of phospho-IRF3 is associated with the EGFR. (j) EGFR associates with IRF3 and TBK1 in the membrane fraction. Membrane, cytosolic or nuclear fractions were prepared from U251EGFR cells. Immunoprecipitation was conducted with IRF3 or TBK1 antibody followed by Western blot with EGFR. E-Cadherin, GAPDH and lamin blots were done to test purity of various fractions. Cells were serum starved and cultured in serum free DMEM overnight for all experiments. The EGF concentration used was 50ng/ml. (k) In vitro transcribed and translated EGFR was incubated with GST-IRF3 or GST alone followed by Western blot with EGFR. (l) shows in vitro transcribed and translated EGFR. V: empty vector.
    Figure Legend Snippet: EGFR forms a signaling complex with IRF3 and TBK1 (a) EGFR co-immunoprecipitates with IRF3 in U251EGFR cells in the absence of EGF. When EGF is added there is a rapid loss of the EGFR-IRF3 association. (b) Similarly, the EGFR coimmunoprecipitates with TBK1 in U251EGFR cells. When EGF is added there is a rapid loss of the EGFR-TBK1 association. (c) TBK1 is associated with IRF3 in U251EGFR cells in the absence of EGF. When EGF is added to cells, there is a rapid decrease in TBK-IRF3 association. The TBK1-IRF3 association is also disrupted when Erlotinib is used suggesting that it is EGFR driven. C refers to control vehicle (PBS). (d) EGFR co-immunoprecipitates with IRF3 in MDAMB468 cells in the absence of EGF. When EGF is added there is a rapid loss of the EGFR-IRF3 association. (e) Similarly, the EGFR coimmunoprecipitates with TBK1 in MDAMB468 cells. When EGF is added there is a rapid loss of the EGFR-TBK1 association. (f) TBK1 is associated with IRF3 in MDAMB468 cells in the absence of EGF. When EGF is added to cells, there is a rapid decrease in TBK-IRF3 association. The TBK1-IRF3 association is also disrupted when Erlotinib is used suggesting that it is EGFR driven. (g) In contradistinction to IRF3 and TBK1, Shc becomes associated with the EGFR only when EGF is added to U251EGFR cells. (h) About 10% of IRF3 is associated with the EGFR. (i) About 5% of phospho-IRF3 is associated with the EGFR. (j) EGFR associates with IRF3 and TBK1 in the membrane fraction. Membrane, cytosolic or nuclear fractions were prepared from U251EGFR cells. Immunoprecipitation was conducted with IRF3 or TBK1 antibody followed by Western blot with EGFR. E-Cadherin, GAPDH and lamin blots were done to test purity of various fractions. Cells were serum starved and cultured in serum free DMEM overnight for all experiments. The EGF concentration used was 50ng/ml. (k) In vitro transcribed and translated EGFR was incubated with GST-IRF3 or GST alone followed by Western blot with EGFR. (l) shows in vitro transcribed and translated EGFR. V: empty vector.

    Techniques Used: Immunoprecipitation, Western Blot, Cell Culture, Concentration Assay, In Vitro, Incubation, Plasmid Preparation

    25) Product Images from "ASCT2 (SLC1A5) is an EGFR-associated protein that can be co-targeted by cetuximab to sensitize cancer cells to ROS-induced apoptosis"

    Article Title: ASCT2 (SLC1A5) is an EGFR-associated protein that can be co-targeted by cetuximab to sensitize cancer cells to ROS-induced apoptosis

    Journal: Cancer letters

    doi: 10.1016/j.canlet.2016.07.020

    Cetuximab sensitizes cancer cells to ROS-elevating agent-induced apoptosis in an EGFR-expression-dependent manner. ( A ) HN5 cells were subjected to knockdown with each of two different EGFR siRNAs or control siRNA for 72 h. During the last 24 h of siRNA transfection, the cells were either untreated or treated with 10 mM DCA, 20 nM cetuximab, or both. Cell lysates were subjected to Western blotting with the indicated antibodies. ( B ) HN5 and FaDu cells were subjected to knockdown of Rab5 or Rab11 with specific siRNAs or control siRNA for 72 h. During the last 24 h of siRNA transfection, the cells were either untreated or treated with 10 mM DCA, 20 nM cetuximab, or both. Cell lysates were subjected to Western blotting with the indicated antibodies.
    Figure Legend Snippet: Cetuximab sensitizes cancer cells to ROS-elevating agent-induced apoptosis in an EGFR-expression-dependent manner. ( A ) HN5 cells were subjected to knockdown with each of two different EGFR siRNAs or control siRNA for 72 h. During the last 24 h of siRNA transfection, the cells were either untreated or treated with 10 mM DCA, 20 nM cetuximab, or both. Cell lysates were subjected to Western blotting with the indicated antibodies. ( B ) HN5 and FaDu cells were subjected to knockdown of Rab5 or Rab11 with specific siRNAs or control siRNA for 72 h. During the last 24 h of siRNA transfection, the cells were either untreated or treated with 10 mM DCA, 20 nM cetuximab, or both. Cell lysates were subjected to Western blotting with the indicated antibodies.

    Techniques Used: Expressing, Transfection, Western Blot

    ASCT2 is physically associated with EGFR. ( A ) HN5 cell lysates were subjected to EGFR immunoprecipitation (IP) with cetuximab or a control antibody, or subjected to ASCT2 immunoprecipitation with an anti-ASCT2 rabbit monoclonal antibody (D7C12, Cell Signaling) or a control rabbit anti-mouse IgG antibody, followed by Western blotting (WB) of the immunoprecipitates with an anti-ASCT2 rabbit polyclonal antibody (H-52, Santa Cruz Biotechnology) and with an anti-EGFR mouse monoclonal antibody (F4, Sigma-Aldrich), respectively. HC, heavy chain. ( B ) ASCT2 co-immunoprecipitation by cetuximab was validated by Western blotting using different ASCT2 (D7C12) and EGFR (D38B1, Cell Signaling) antibodies as indicated. ( C ) HN5 cells were untransfected or transfected with control siRNA, ASCT2 siRNA, or EGFR siRNA for 72 h before being plated on coverslips for overnight. The cells were then incubated with blocking buffer only or with an ASCT2 antibody (H-52) and an EGFR antibody (F4), alone and together, as indicated, and then subjected to Duolink proximity ligation assay as described in Materials and methods. Scale bars, 25 μm. ( D ) Lysates from the indicated HNSCC cell lines were subjected to EGFR immunoprecipitation with cetuximab or ASCT2 immunoprecipitation with an ASCT2 antibody (H-52), along with a control IgG immunoprecipitation. The immunoprecipitates were then analyzed by Western blotting with the indicated antibodies.
    Figure Legend Snippet: ASCT2 is physically associated with EGFR. ( A ) HN5 cell lysates were subjected to EGFR immunoprecipitation (IP) with cetuximab or a control antibody, or subjected to ASCT2 immunoprecipitation with an anti-ASCT2 rabbit monoclonal antibody (D7C12, Cell Signaling) or a control rabbit anti-mouse IgG antibody, followed by Western blotting (WB) of the immunoprecipitates with an anti-ASCT2 rabbit polyclonal antibody (H-52, Santa Cruz Biotechnology) and with an anti-EGFR mouse monoclonal antibody (F4, Sigma-Aldrich), respectively. HC, heavy chain. ( B ) ASCT2 co-immunoprecipitation by cetuximab was validated by Western blotting using different ASCT2 (D7C12) and EGFR (D38B1, Cell Signaling) antibodies as indicated. ( C ) HN5 cells were untransfected or transfected with control siRNA, ASCT2 siRNA, or EGFR siRNA for 72 h before being plated on coverslips for overnight. The cells were then incubated with blocking buffer only or with an ASCT2 antibody (H-52) and an EGFR antibody (F4), alone and together, as indicated, and then subjected to Duolink proximity ligation assay as described in Materials and methods. Scale bars, 25 μm. ( D ) Lysates from the indicated HNSCC cell lines were subjected to EGFR immunoprecipitation with cetuximab or ASCT2 immunoprecipitation with an ASCT2 antibody (H-52), along with a control IgG immunoprecipitation. The immunoprecipitates were then analyzed by Western blotting with the indicated antibodies.

    Techniques Used: Immunoprecipitation, Western Blot, Transfection, Incubation, Blocking Assay, Proximity Ligation Assay

    26) Product Images from "Absence of miR-146a in Podocytes Increases Risk of Diabetic Glomerulopathy via Up-regulation of ErbB4 and Notch-1 *"

    Article Title: Absence of miR-146a in Podocytes Increases Risk of Diabetic Glomerulopathy via Up-regulation of ErbB4 and Notch-1 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.753822

    Working model. A diagram showing our working model. Under homeostatic conditions of normoglycemia, podocyte expressed miR-146a suppresses expression of Notch-1 and ErbB4 to maintain healthy cells. Diabetic milieu , including TGF-β1 (and MCP-1), induces MCP1 and MCPIP1 in podocytes, which decreases miR-146a levels. This results in de-repression of Notch-1 and ErbB4, which together with EGFR, induce podocyte injury. Blocking this signaling pathway with ErbB4/EGFR inhibitors suppresses the harmful signaling and decreases podocyte injury in vitro and glomerulopathy in vivo .
    Figure Legend Snippet: Working model. A diagram showing our working model. Under homeostatic conditions of normoglycemia, podocyte expressed miR-146a suppresses expression of Notch-1 and ErbB4 to maintain healthy cells. Diabetic milieu , including TGF-β1 (and MCP-1), induces MCP1 and MCPIP1 in podocytes, which decreases miR-146a levels. This results in de-repression of Notch-1 and ErbB4, which together with EGFR, induce podocyte injury. Blocking this signaling pathway with ErbB4/EGFR inhibitors suppresses the harmful signaling and decreases podocyte injury in vitro and glomerulopathy in vivo .

    Techniques Used: Expressing, Blocking Assay, In Vitro, In Vivo

    STZ treatment of WT and miR-146a −/− mice results in increased glomerular injury and induction of miR-146a targets in the mouse glomeruli that is suppressed by erlotinib. A, STZ-induced up-regulation of EGFR, Notch-1, and ErbB4 expression in the mouse glomeruli is reduced by erlotinib. Representative confocal microscopy images of immunofluorescently labeled glomeruli from WT ( top panels ) and miR-146a −/− ( bottom panels ) mice treated with vehicle alone ( Control ), with STZ and vehicle ( STZ ) or with STZ and erlotinib ( STZ + Erl ). Kidney sections were imaged after staining with DAPI and antibodies against EGFR, Notch-1, ErbB4, and Synaptopodin ( Synpo ). Merged DAPI, EGFR and Synpo, DAPI, Notch-1 and Synpo, and DAPI, ErbB4 and Synpo channels are also presented that show podocyte colocalization for these proteins. Scale bar , 50 μm. B , graphs showing the quantification of relative glomerular signal intensity of EGFR, Notch-1, and ErbB4 in samples from A . Data shown are mean ± S.E. ( n = 5/group). ns , not significant; **, p
    Figure Legend Snippet: STZ treatment of WT and miR-146a −/− mice results in increased glomerular injury and induction of miR-146a targets in the mouse glomeruli that is suppressed by erlotinib. A, STZ-induced up-regulation of EGFR, Notch-1, and ErbB4 expression in the mouse glomeruli is reduced by erlotinib. Representative confocal microscopy images of immunofluorescently labeled glomeruli from WT ( top panels ) and miR-146a −/− ( bottom panels ) mice treated with vehicle alone ( Control ), with STZ and vehicle ( STZ ) or with STZ and erlotinib ( STZ + Erl ). Kidney sections were imaged after staining with DAPI and antibodies against EGFR, Notch-1, ErbB4, and Synaptopodin ( Synpo ). Merged DAPI, EGFR and Synpo, DAPI, Notch-1 and Synpo, and DAPI, ErbB4 and Synpo channels are also presented that show podocyte colocalization for these proteins. Scale bar , 50 μm. B , graphs showing the quantification of relative glomerular signal intensity of EGFR, Notch-1, and ErbB4 in samples from A . Data shown are mean ± S.E. ( n = 5/group). ns , not significant; **, p

    Techniques Used: Mouse Assay, Expressing, Confocal Microscopy, Labeling, Staining

    27) Product Images from "Two mature products of MIR-491 coordinate to suppress key cancer hallmarks in glioblastoma"

    Article Title: Two mature products of MIR-491 coordinate to suppress key cancer hallmarks in glioblastoma

    Journal: Oncogene

    doi: 10.1038/onc.2014.98

    miR-491-5p and/or miR-491-3p directly target IGFBP2, CDK6, EGFR, and Bcl-xL and regulate GBM cell proliferation (a) miR-491-5p/miR-491-3p regulates IGFBP2, CDK6, EGFR, and Bcl-xL expression at the protein level. Beta-actin was used as a protein loading control. Each band's intensity was quantified by using Image J, and the relative values (beta-actin as internal control) were shown below the bands. +: 25nM; ++: 50 nM. (b) miR-491 inhibits glioma cell growth. Cell viability of U251 and T98G cells transfected with both miR-491-5p and miR-491-3p mimics or with control mimics was monitored by MTT assay (n = 6). (c) miR-491 inhibits soft agar colonization by glioma cell lines. Upon transfection, cells were seeded into the soft agar and the numbers of colonies were determined four weeks later (n = 3); representative colony morphologies of U251 are shown (n = 3). Bar: 200 μm. (d) miR-491 inhibits glioma cell proliferation. BrdU incorporation assay (n=3) was done seventy-two hours after transfection. Data are presented as mean ± SD (*, P
    Figure Legend Snippet: miR-491-5p and/or miR-491-3p directly target IGFBP2, CDK6, EGFR, and Bcl-xL and regulate GBM cell proliferation (a) miR-491-5p/miR-491-3p regulates IGFBP2, CDK6, EGFR, and Bcl-xL expression at the protein level. Beta-actin was used as a protein loading control. Each band's intensity was quantified by using Image J, and the relative values (beta-actin as internal control) were shown below the bands. +: 25nM; ++: 50 nM. (b) miR-491 inhibits glioma cell growth. Cell viability of U251 and T98G cells transfected with both miR-491-5p and miR-491-3p mimics or with control mimics was monitored by MTT assay (n = 6). (c) miR-491 inhibits soft agar colonization by glioma cell lines. Upon transfection, cells were seeded into the soft agar and the numbers of colonies were determined four weeks later (n = 3); representative colony morphologies of U251 are shown (n = 3). Bar: 200 μm. (d) miR-491 inhibits glioma cell proliferation. BrdU incorporation assay (n=3) was done seventy-two hours after transfection. Data are presented as mean ± SD (*, P

    Techniques Used: Expressing, Transfection, MTT Assay, BrdU Incorporation Assay

    Knockdown of miR-491-5p exacerbates malignancy of Ink4a-Arf -null mouse glial progenitor cells (a) Knockdown efficiency of anti-miR-491-5p (hairpin inhibitor). Forty-eight hours after transfection with anti-miR-491-5p, miR-491-5p levels were assessed by quantitative PCR. (b) Anti-miR-491-5p upregulates mouse CDK6, EGFR, and Bcl-xL expression. Beta-actin was used as a protein loading control. Number below each blot indicates relative band intensity (quantified by Image J). (c) Anti-miR-491-5p promotes the growth of Ink4a-Arf -null mouse glial progenitor cells. Cell viability was monitored by MTT assay (n = 6). (d) Anti-miR-491 enhances cell proliferation. Proliferating cells were analyzed with BrdU incorporation assay seventy-two hours after transfection. (e) Anti-miR-491 promotes cell invasion. Invading cells were counted in ten randomly chosen fields under the microscope, with the representative photographs taken at 100 × magnification (n = 3). Data are presented as mean ± SD (*, P
    Figure Legend Snippet: Knockdown of miR-491-5p exacerbates malignancy of Ink4a-Arf -null mouse glial progenitor cells (a) Knockdown efficiency of anti-miR-491-5p (hairpin inhibitor). Forty-eight hours after transfection with anti-miR-491-5p, miR-491-5p levels were assessed by quantitative PCR. (b) Anti-miR-491-5p upregulates mouse CDK6, EGFR, and Bcl-xL expression. Beta-actin was used as a protein loading control. Number below each blot indicates relative band intensity (quantified by Image J). (c) Anti-miR-491-5p promotes the growth of Ink4a-Arf -null mouse glial progenitor cells. Cell viability was monitored by MTT assay (n = 6). (d) Anti-miR-491 enhances cell proliferation. Proliferating cells were analyzed with BrdU incorporation assay seventy-two hours after transfection. (e) Anti-miR-491 promotes cell invasion. Invading cells were counted in ten randomly chosen fields under the microscope, with the representative photographs taken at 100 × magnification (n = 3). Data are presented as mean ± SD (*, P

    Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Expressing, MTT Assay, BrdU Incorporation Assay, Microscopy

    miR-491-5p and -3p are downregulated in GBM and their expression inversely correlated with that of IGFBP2 , EGFR , CDK6 , and Bcl-xL (a) Deletion of 9p21.3 is frequent in TCGA GBM data. Segmented DNA copy-number data from SNP arrays are shown. Each row represents a patient; deleted regions are shown in blue. (b) Chromosomal copy number variations (CNV) of both CDKN2A and MIR-491 in different cancer types from TCGA. GBM, glioblastoma multiforme; LUSC, Lung squamous cell carcinoma; OvCa, Ovarian serous cystadenocarcinoma; BRCA, Breast invasive carcinoma; UCEC, Uterine corpus endometrioid carcinoma; CRC, Colon and rectum adenocarcinoma; BLCA, Bladder urothelial carcinoma. (c) Genomic loss (del) leads to a significant decrease in miR-491-5p expression (p
    Figure Legend Snippet: miR-491-5p and -3p are downregulated in GBM and their expression inversely correlated with that of IGFBP2 , EGFR , CDK6 , and Bcl-xL (a) Deletion of 9p21.3 is frequent in TCGA GBM data. Segmented DNA copy-number data from SNP arrays are shown. Each row represents a patient; deleted regions are shown in blue. (b) Chromosomal copy number variations (CNV) of both CDKN2A and MIR-491 in different cancer types from TCGA. GBM, glioblastoma multiforme; LUSC, Lung squamous cell carcinoma; OvCa, Ovarian serous cystadenocarcinoma; BRCA, Breast invasive carcinoma; UCEC, Uterine corpus endometrioid carcinoma; CRC, Colon and rectum adenocarcinoma; BLCA, Bladder urothelial carcinoma. (c) Genomic loss (del) leads to a significant decrease in miR-491-5p expression (p

    Techniques Used: Expressing

    miR-491 inhibits GSC propagation in vitro and in vivo (a) miR-491 inhibits neurosphere formation of GSC 11. Patient-derived GSC 11 upon transfection were cultured in NSC medium, and the number of spheres and their diameters were determined 7 days later; representative sphere morphologies are shown (n = 3). Bar: 100 μm. (b) miR-491 inhibits self-renewal of GSC 11. Transfected GSC11 were cultured in NSC medium to form neurospheres and serially passaged every 7 days. The number of primary, secondary, and tertiary neurospheres were determined respectively (n = 3). (c) miR-491 inhibits GSC proliferation.GSC11 cells were subjected to BrdU incorporation assay seventy-two hours after transfection. (d) miR-491 induces GSC apoptosis. Apoptotic cells were identified by APO-BrdU kit seventy-two hours after transfection; PARP cleavage was determined by Western blot (in f). (e, f) miR-491 overexpression leads to reduction of EGFR, CDK6, IGFBP2, and Bcl-xL (e), reduction of p-Akt, Nestin, and Sox2 (f), as well as upregulation of GFAP in GSC11 cells (f). (g, h) IGFBP2, CDK6, or EGFR overexpression partially overcomes the inhibitory effect of miR-491 on neurosphere formation. GSC 11 cells were transfected as described in Methods. IGFBP2, CDK6, EGFR and Bcl-xL levels were analyzed by Western blot 48 hours after transfection (g) and the number of spheres and their diameters were determined 7 days later (h) (n = 3). In a-d and h, data are presented as mean ± SD (*, P
    Figure Legend Snippet: miR-491 inhibits GSC propagation in vitro and in vivo (a) miR-491 inhibits neurosphere formation of GSC 11. Patient-derived GSC 11 upon transfection were cultured in NSC medium, and the number of spheres and their diameters were determined 7 days later; representative sphere morphologies are shown (n = 3). Bar: 100 μm. (b) miR-491 inhibits self-renewal of GSC 11. Transfected GSC11 were cultured in NSC medium to form neurospheres and serially passaged every 7 days. The number of primary, secondary, and tertiary neurospheres were determined respectively (n = 3). (c) miR-491 inhibits GSC proliferation.GSC11 cells were subjected to BrdU incorporation assay seventy-two hours after transfection. (d) miR-491 induces GSC apoptosis. Apoptotic cells were identified by APO-BrdU kit seventy-two hours after transfection; PARP cleavage was determined by Western blot (in f). (e, f) miR-491 overexpression leads to reduction of EGFR, CDK6, IGFBP2, and Bcl-xL (e), reduction of p-Akt, Nestin, and Sox2 (f), as well as upregulation of GFAP in GSC11 cells (f). (g, h) IGFBP2, CDK6, or EGFR overexpression partially overcomes the inhibitory effect of miR-491 on neurosphere formation. GSC 11 cells were transfected as described in Methods. IGFBP2, CDK6, EGFR and Bcl-xL levels were analyzed by Western blot 48 hours after transfection (g) and the number of spheres and their diameters were determined 7 days later (h) (n = 3). In a-d and h, data are presented as mean ± SD (*, P

    Techniques Used: In Vitro, In Vivo, Derivative Assay, Transfection, Cell Culture, BrdU Incorporation Assay, Western Blot, Over Expression

    28) Product Images from "EGF Promotes the Shedding of Soluble E-cadherin in an ADAM10-dependent Manner in Prostate Epithelial Cells"

    Article Title: EGF Promotes the Shedding of Soluble E-cadherin in an ADAM10-dependent Manner in Prostate Epithelial Cells

    Journal: Cellular signalling

    doi: 10.1016/j.cellsig.2011.10.004

    EGFR ligands promote the generation of sE-cadherin in an ADAM10-dependent manner
    Figure Legend Snippet: EGFR ligands promote the generation of sE-cadherin in an ADAM10-dependent manner

    Techniques Used:

    29) Product Images from "Identification of Human Fibroblast Cell Lines as a Feeder Layer for Human Corneal Epithelial Regeneration"

    Article Title: Identification of Human Fibroblast Cell Lines as a Feeder Layer for Human Corneal Epithelial Regeneration

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038825

    Representative immuno-fluorescent staining profiles for corneal epithelial phenotype. Corneal epithelial markers, including the differentiation markers, keratin 3 (K3), and connexin 43 (Cx43), as well as progenitor markers, EGFR, nuclear p63 and integrin β1, were expressed by HLE regenerated on human feeder of Hs68 fibroblasts; Hoechst 33342 was used for nuclear counterstaining.
    Figure Legend Snippet: Representative immuno-fluorescent staining profiles for corneal epithelial phenotype. Corneal epithelial markers, including the differentiation markers, keratin 3 (K3), and connexin 43 (Cx43), as well as progenitor markers, EGFR, nuclear p63 and integrin β1, were expressed by HLE regenerated on human feeder of Hs68 fibroblasts; Hoechst 33342 was used for nuclear counterstaining.

    Techniques Used: Staining

    30) Product Images from "ΕGFR/ERβ-Mediated Cell Morphology and Invasion Capacity Are Associated with Matrix Culture Substrates in Breast Cancer"

    Article Title: ΕGFR/ERβ-Mediated Cell Morphology and Invasion Capacity Are Associated with Matrix Culture Substrates in Breast Cancer

    Journal: Cells

    doi: 10.3390/cells9102256

    EGFR/ERβ axis regulates functional properties of MDA-MB-231 and shERβ MDA-MB-231 breast cancer cells. ( A ) Collagen type I adhesion. ( B ) Cell invasion in collagen type I matrix. Treatments with AG1478 (2 μM) and/or E2 (10 nM) were performed for 24 h prior in MDA-MB-231 and shERβ MDA-MB-231 cells. Each bar represents mean ± SD values from triplicate samples. Statistically significant differences are indicated accordingly: * p
    Figure Legend Snippet: EGFR/ERβ axis regulates functional properties of MDA-MB-231 and shERβ MDA-MB-231 breast cancer cells. ( A ) Collagen type I adhesion. ( B ) Cell invasion in collagen type I matrix. Treatments with AG1478 (2 μM) and/or E2 (10 nM) were performed for 24 h prior in MDA-MB-231 and shERβ MDA-MB-231 cells. Each bar represents mean ± SD values from triplicate samples. Statistically significant differences are indicated accordingly: * p

    Techniques Used: Functional Assay, Multiple Displacement Amplification

    EGFR inhibition affects the expression and activity levels of proteases in MDA-MB-231 and shERβ MDA-MB-231 breast cancer cells. ( A ) Quantitative RT-PCR analysis of MMP7 and MT1-MMP mRNA levels after 24 h without and with treatments (AG1478, E2 and mix). ( B , C ) MMP2/MMP9 gelatinolytic activities (as assayed by gelatin zymography) in MDA-MB-231 and shERβ MDA-MB-231 cells, before and after treatments (AG1478, E2, mix and 24 h). Each bar represents mean ± SD values from triplicate samples. Statistically significant differences are indicated accordingly: * p
    Figure Legend Snippet: EGFR inhibition affects the expression and activity levels of proteases in MDA-MB-231 and shERβ MDA-MB-231 breast cancer cells. ( A ) Quantitative RT-PCR analysis of MMP7 and MT1-MMP mRNA levels after 24 h without and with treatments (AG1478, E2 and mix). ( B , C ) MMP2/MMP9 gelatinolytic activities (as assayed by gelatin zymography) in MDA-MB-231 and shERβ MDA-MB-231 cells, before and after treatments (AG1478, E2, mix and 24 h). Each bar represents mean ± SD values from triplicate samples. Statistically significant differences are indicated accordingly: * p

    Techniques Used: Inhibition, Expressing, Activity Assay, Multiple Displacement Amplification, Quantitative RT-PCR, Zymography

    31) Product Images from "Anoikis evasion in inflammatory breast cancer cells is mediated by Bim-EL sequestration"

    Article Title: Anoikis evasion in inflammatory breast cancer cells is mediated by Bim-EL sequestration

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2014.209

    ErbB2 and EGFR are necessary for anoikis protection in KPL-4 and SUM149 cells, respectively. ( a ) KPL-4 cells were transduced with a lentivirus containing either an empty vector (EV) or an ErbB2 shRNA-containing vector (shErbB2), and western blotting analysis
    Figure Legend Snippet: ErbB2 and EGFR are necessary for anoikis protection in KPL-4 and SUM149 cells, respectively. ( a ) KPL-4 cells were transduced with a lentivirus containing either an empty vector (EV) or an ErbB2 shRNA-containing vector (shErbB2), and western blotting analysis

    Techniques Used: Transduction, Plasmid Preparation, shRNA, Western Blot

    32) Product Images from "RYBP Inhibits Progression and Metastasis of Lung Cancer by Suppressing EGFR Signaling and Epithelial-Mesenchymal Transition"

    Article Title: RYBP Inhibits Progression and Metastasis of Lung Cancer by Suppressing EGFR Signaling and Epithelial-Mesenchymal Transition

    Journal: Translational Oncology

    doi: 10.1016/j.tranon.2017.01.004

    RYBP regulated LC progression via the EGFR/AKT/ERK/EMT signaling pathway. (A) Knockdown of RYBP expression upregulated EGFR/AKT/ERK expression and EMT signaling pathways in A549 cells. (B) Overexpression of RYBP expression attenuated EGFR/AKT/ERK expression and the EMT signal pathway in PC9 cells. GAPDH was loaded as the control.
    Figure Legend Snippet: RYBP regulated LC progression via the EGFR/AKT/ERK/EMT signaling pathway. (A) Knockdown of RYBP expression upregulated EGFR/AKT/ERK expression and EMT signaling pathways in A549 cells. (B) Overexpression of RYBP expression attenuated EGFR/AKT/ERK expression and the EMT signal pathway in PC9 cells. GAPDH was loaded as the control.

    Techniques Used: Expressing, Over Expression

    33) Product Images from "Proteomics reveals novel protein associations with early endosomes in an epidermal growth factor–dependent manner"

    Article Title: Proteomics reveals novel protein associations with early endosomes in an epidermal growth factor–dependent manner

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA117.000632

    Total and phosphorylated EGFR colocalize with early endosomal markers following isotonic Percoll gradient fractionation. A , PNS was prepared from HeLa cells treated with and without EGF (10 ng/ml) for 15 min. PNS was resolved on a 17% isotonic Percoll gradient, fractionated, and resolved by 7.5% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and immunoblotted for phosphorylated (Tyr(P)-1068, pY1068 ) and total EGFR as well as the following marker proteins: EEA1 (early endosomes), TfnR (early and recycling endosomes), LAMP2 (late endosomes and lysosomes), Na/K-ATPase (plasma membrane), and Calnexin (endoplasmic reticulum). The immunoblots are representative of three independent experiments. B , relative intensity of the immunoblots in A. Circles on the x axis represent density bead migration ( R f ∼0.93 = 1.109 g/ml, ∼0.91 = 1.070 g/ml, ∼0.89 = 1.057 g/ml, ∼0.59 = 1.049 g/ml, and ∼0.20 = 1.042 g/ml).
    Figure Legend Snippet: Total and phosphorylated EGFR colocalize with early endosomal markers following isotonic Percoll gradient fractionation. A , PNS was prepared from HeLa cells treated with and without EGF (10 ng/ml) for 15 min. PNS was resolved on a 17% isotonic Percoll gradient, fractionated, and resolved by 7.5% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and immunoblotted for phosphorylated (Tyr(P)-1068, pY1068 ) and total EGFR as well as the following marker proteins: EEA1 (early endosomes), TfnR (early and recycling endosomes), LAMP2 (late endosomes and lysosomes), Na/K-ATPase (plasma membrane), and Calnexin (endoplasmic reticulum). The immunoblots are representative of three independent experiments. B , relative intensity of the immunoblots in A. Circles on the x axis represent density bead migration ( R f ∼0.93 = 1.109 g/ml, ∼0.91 = 1.070 g/ml, ∼0.89 = 1.057 g/ml, ∼0.59 = 1.049 g/ml, and ∼0.20 = 1.042 g/ml).

    Techniques Used: Fractionation, SDS Page, Marker, Western Blot, Migration

    EGF and EGFR colocalization with EEA1 in siCON, RUFY1 KD, and PTPN23 KD cells. A–C , HeLa cells were incubated with siCON, RUFY1, or PTPN23 siRNA for 72 h prior to serum starving. The serum-starved cells were pulse-labeled with 10 ng/ml Alexa Fluor 647–EGF ligand (Invitrogen) for 0, 15, 30, 60, and 120 min, followed by fixation in 4% paraformaldehyde. The cells were permeabilized and immunostained for EEA1 and EGFR and visualized using either a goat anti-rabbit Alexa 488 or goat anti-mouse Alexa 568, respectively. Images are representative of time points from three independent experiments. The extent of colocalization between EGF or EGFR and EEA1 was measured as described under “Experimental procedures.” The data are plotted as the percentages of colocalization for each time point. Approximately 300 cells were analyzed per time point per condition, per experiment. Scale bars , 20 μm. Images were quantified using ImageJ software. D , a representative immunoblot from each knockdown experiment, probing for PTPN23, RUFY1, and α-tubulin. For each knockdown experiment, the samples were loaded in multiple protein concentrations (20, 10, and 5 μg), and the percentage of knockdown was calculated. Only experiments with a knockdown efficiency of > 90% were used.
    Figure Legend Snippet: EGF and EGFR colocalization with EEA1 in siCON, RUFY1 KD, and PTPN23 KD cells. A–C , HeLa cells were incubated with siCON, RUFY1, or PTPN23 siRNA for 72 h prior to serum starving. The serum-starved cells were pulse-labeled with 10 ng/ml Alexa Fluor 647–EGF ligand (Invitrogen) for 0, 15, 30, 60, and 120 min, followed by fixation in 4% paraformaldehyde. The cells were permeabilized and immunostained for EEA1 and EGFR and visualized using either a goat anti-rabbit Alexa 488 or goat anti-mouse Alexa 568, respectively. Images are representative of time points from three independent experiments. The extent of colocalization between EGF or EGFR and EEA1 was measured as described under “Experimental procedures.” The data are plotted as the percentages of colocalization for each time point. Approximately 300 cells were analyzed per time point per condition, per experiment. Scale bars , 20 μm. Images were quantified using ImageJ software. D , a representative immunoblot from each knockdown experiment, probing for PTPN23, RUFY1, and α-tubulin. For each knockdown experiment, the samples were loaded in multiple protein concentrations (20, 10, and 5 μg), and the percentage of knockdown was calculated. Only experiments with a knockdown efficiency of > 90% were used.

    Techniques Used: Incubation, Labeling, Software

    34) Product Images from "Heparin Binding Epidermal Growth Factor-Like Growth Factor and PD169316 Prevent Apoptosis in Mouse Embryonic Stem Cells"

    Article Title: Heparin Binding Epidermal Growth Factor-Like Growth Factor and PD169316 Prevent Apoptosis in Mouse Embryonic Stem Cells

    Journal:

    doi: 10.1093/jb/mvn153

    Identification of EGFR phosphorylation sites involved in anti-CD9-induced apoptosis (20×). Twenty-four hrs of anti-CD9 antibody treatment was performed, cells were fixed and stained for activated EGFR Y845, 1068, 1086, 1148 and 1173 (green). Nuclei
    Figure Legend Snippet: Identification of EGFR phosphorylation sites involved in anti-CD9-induced apoptosis (20×). Twenty-four hrs of anti-CD9 antibody treatment was performed, cells were fixed and stained for activated EGFR Y845, 1068, 1086, 1148 and 1173 (green). Nuclei

    Techniques Used: Staining

    Phosphorylation of EGFR is suppressed by HB-EGF and p38 MAPK inhibitor (20×). After 24 h of anti-CD9 antibody treatment and fixation cells were stained for activated EGFR 1148 and 1173 (green). Nuclei are labelled with DAPI. Anti-CD9 antibody
    Figure Legend Snippet: Phosphorylation of EGFR is suppressed by HB-EGF and p38 MAPK inhibitor (20×). After 24 h of anti-CD9 antibody treatment and fixation cells were stained for activated EGFR 1148 and 1173 (green). Nuclei are labelled with DAPI. Anti-CD9 antibody

    Techniques Used: Staining

    35) Product Images from "Dacomitinib, a pan-inhibitor of ErbB receptors, suppresses growth and invasive capacity of chemoresistant ovarian carcinoma cells"

    Article Title: Dacomitinib, a pan-inhibitor of ErbB receptors, suppresses growth and invasive capacity of chemoresistant ovarian carcinoma cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-04147-0

    The ErbB family contributes to cisplatin resistance. ( A ) Correlation of expression of HRG1 - α , HRG1 - β , EGFR and HER2 with resistance to cisplatin. EOC cell lines with higher expression of HRG1 - α , HRG1 - β , EGFR and HER2 showed significantly higher cisplatin IC 50 values. The correlation coefficient (r) between the expression of EGFR and HER2 and cisplatin concentrations was 0.917 ( P = 0.0281) and 0.890 ( P = 0.0341), respectively. ( B ) Dacomitinib inhibits ErbB activation. The effect of dacomitinib (5 μM) on ErbB activation was determined by Western blot analysis. Protein lysates were subjected to Western blotting and probed with the indicated antibodies. β-actin was used as the loading control. The blots are representative of three independent experiments with similar results. ( C ) The effects of the ErbB inhibitors on HRGβ-1-induced proliferation in cisplatin-treated Caov4 cells were shown by MTT assay. The cells were pre-treated with the anti-ErbB agents for 4 h, followed by treatment with HRGβ-1 for 48 h. ( D ) The effects of the ErbB inhibitors-cisplatin therapy on cell proliferation were investigated by MTT assay after 48 h of treatment and the data are shown by IC 50 shift analysis. The concentrations of cisplatin were 0.1, 0.5, 1, 2.5, 5 and 10 μg/mL. ( E ) Normalised isobolograms of combination of erlotinib (5 μM) and dacomitinib (5 μM) with cisplatin. The data were analysed using the CalcuSyn software. The connecting line represents additivity. Data points located below the line indicate a synergistic drug-drug interaction and data points above the line indicate an antagonistic interaction. The numbers under the isobolograms indicate the concentrations of the drugs in combination. Data shown represent the mean ± SD from three independent experiments, each performed in triplicate. Statistically significant values of * p
    Figure Legend Snippet: The ErbB family contributes to cisplatin resistance. ( A ) Correlation of expression of HRG1 - α , HRG1 - β , EGFR and HER2 with resistance to cisplatin. EOC cell lines with higher expression of HRG1 - α , HRG1 - β , EGFR and HER2 showed significantly higher cisplatin IC 50 values. The correlation coefficient (r) between the expression of EGFR and HER2 and cisplatin concentrations was 0.917 ( P = 0.0281) and 0.890 ( P = 0.0341), respectively. ( B ) Dacomitinib inhibits ErbB activation. The effect of dacomitinib (5 μM) on ErbB activation was determined by Western blot analysis. Protein lysates were subjected to Western blotting and probed with the indicated antibodies. β-actin was used as the loading control. The blots are representative of three independent experiments with similar results. ( C ) The effects of the ErbB inhibitors on HRGβ-1-induced proliferation in cisplatin-treated Caov4 cells were shown by MTT assay. The cells were pre-treated with the anti-ErbB agents for 4 h, followed by treatment with HRGβ-1 for 48 h. ( D ) The effects of the ErbB inhibitors-cisplatin therapy on cell proliferation were investigated by MTT assay after 48 h of treatment and the data are shown by IC 50 shift analysis. The concentrations of cisplatin were 0.1, 0.5, 1, 2.5, 5 and 10 μg/mL. ( E ) Normalised isobolograms of combination of erlotinib (5 μM) and dacomitinib (5 μM) with cisplatin. The data were analysed using the CalcuSyn software. The connecting line represents additivity. Data points located below the line indicate a synergistic drug-drug interaction and data points above the line indicate an antagonistic interaction. The numbers under the isobolograms indicate the concentrations of the drugs in combination. Data shown represent the mean ± SD from three independent experiments, each performed in triplicate. Statistically significant values of * p

    Techniques Used: Expressing, Activation Assay, Western Blot, MTT Assay, Software

    Related Articles

    Isolation:

    Article Title: Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells, et al. Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells
    Article Snippet: .. 2.3 qRT‐PCR analysis of gene expression To measure the expression level of EGFR and MET in obtained cell lines, total RNA was isolated using GenElute™ Mammalian Total RNA Miniprep Kit (Sigma‐Aldrich) following the manufacturer's protocol. .. After DNase I (Sigma‐Aldrich) treatment, reverse transcription reaction was performed using 0.5 μg of RNA and the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following the manufacturer's instructions.

    Labeling:

    Article Title: Epidermal Growth Factor Receptor Activation by Epidermal Growth Factor Mediates Oxidant-Induced Goblet Cell Metaplasia in Human Airway Epithelium
    Article Snippet: .. After blocking as described previously, the cells were labeled with anti-10 μg/ml EGFR (Ab-3; Calbiochem), 20 μg/ml anti-Bcl-2 (Chemicon), or anti-LPO (1:200, generated as described) ( ) antibodies. ..

    Generated:

    Article Title: Epidermal Growth Factor Receptor Activation by Epidermal Growth Factor Mediates Oxidant-Induced Goblet Cell Metaplasia in Human Airway Epithelium
    Article Snippet: .. After blocking as described previously, the cells were labeled with anti-10 μg/ml EGFR (Ab-3; Calbiochem), 20 μg/ml anti-Bcl-2 (Chemicon), or anti-LPO (1:200, generated as described) ( ) antibodies. ..

    other:

    Article Title: The ErbB2ΔEx16 splice variant is a major oncogenic driver in breast cancer that promotes a pro-metastatic tumor microenvironment
    Article Snippet: Antibodies The following primary antibodies were used for immunoblotting: (Santa Cruz Biotechnology) ErbB3 C17 (sc-285), (Cell Signaling Technology) phospho-ErbB2 Y1221/1222 (2249), EGFR (2232), phospho-Src Family Kinase Y416 (2101), phospho-ErbB3 Y1289 (4791), phospho-p38 (9215), p38 T180/Y182 (9212), tubulin (2148), phospho-Smad2 S245/250/255 (3104), Smad2/3 (3102), HIF1α (3716), phospho-Stat3 Y705 (9145), Stat3 (9139), phospho-YB1 S102 (2900), (Millipore) c-ErbB2 Ab-3 (OP-15), (BD Transduction) E-cadherin (610182), (Abcam) YB1 (12148).

    Blocking Assay:

    Article Title: Epidermal Growth Factor Receptor Activation by Epidermal Growth Factor Mediates Oxidant-Induced Goblet Cell Metaplasia in Human Airway Epithelium
    Article Snippet: .. After blocking as described previously, the cells were labeled with anti-10 μg/ml EGFR (Ab-3; Calbiochem), 20 μg/ml anti-Bcl-2 (Chemicon), or anti-LPO (1:200, generated as described) ( ) antibodies. ..

    Expressing:

    Article Title: 89Zr-Onartuzumab PET imaging of c-MET receptor dynamics
    Article Snippet: .. Cetuximab (5 mg/mL; Merck) and onartuzumab (60 mg/mL; Genentech) served as primary antibodies for EGFR and c-MET, respectively, and mouse anti-human Fc-specific FITC-conjugated secondary antibody (clone HP-6017; Sigma-Aldrich) was used for readout of both EGFR and c-MET expression, with 10,000 events assessed per sample. .. The sensitivity of HCC827 and HCC827ErlRes cell lines to erlotinib and NVP-AUY-922 after 4 days of treatment was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [ ].

    Article Title: Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells, et al. Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells
    Article Snippet: .. 2.3 qRT‐PCR analysis of gene expression To measure the expression level of EGFR and MET in obtained cell lines, total RNA was isolated using GenElute™ Mammalian Total RNA Miniprep Kit (Sigma‐Aldrich) following the manufacturer's protocol. .. After DNase I (Sigma‐Aldrich) treatment, reverse transcription reaction was performed using 0.5 μg of RNA and the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following the manufacturer's instructions.

    Western Blot:

    Article Title: DDX3X Induces Primary EGFR-TKI Resistance Based on Intratumor Heterogeneity in Lung Cancer Cells Harboring EGFR-Activating Mutations
    Article Snippet: .. Immunoblots from tumor cell lysates were probed with antibodies against DDX3X (Sigma), EGFR, phospho-EGFR (Tyr1068), phospho-EGFR (Tyr1173), phospho-EGFR (Tyr845), Akt, phospho-Akt, Erk1/2, phospho-Erk1/2, and β-actin (Sigma). .. All antibodies except for anti-DDX3X and anti-β-actin were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA).

    Quantitative RT-PCR:

    Article Title: Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells, et al. Expression level of EGFR and MET receptors regulates invasiveness of melanoma cells
    Article Snippet: .. 2.3 qRT‐PCR analysis of gene expression To measure the expression level of EGFR and MET in obtained cell lines, total RNA was isolated using GenElute™ Mammalian Total RNA Miniprep Kit (Sigma‐Aldrich) following the manufacturer's protocol. .. After DNase I (Sigma‐Aldrich) treatment, reverse transcription reaction was performed using 0.5 μg of RNA and the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following the manufacturer's instructions.

    Binding Assay:

    Article Title: ATP-mediated Activation of the NADPH Oxidase DUOX1 Mediates Airway Epithelial Responses to Bacterial Stimuli *
    Article Snippet: .. To avoid TGF-α binding to EGFR, cells were in some cases pretreated with an α-EGFR mAb (225; 4 μg/ml; Calbiochem) for 30 min prior to stimulation. .. Upon starvation, cell monolayers were stimulated in the presence of 10 μ m of fluorogenic ADAM17 Substrate II (Calbiochem) for 2 h, after which media were collected.

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  • 91
    Millipore egfr inhibitor
    Inhibition of <t>EGFR-PI3K-Akt</t> pathway attenuates WST-8 reduction. (A~E) Increasing amount of inhibitors was used treat HEK293 cells for 6 hours. Absorbance of WST-8 formazan was recorded after incubation for 1 hour. Data of EGFR inhibitor (A), PI3K inhibitor <t>LY294002</t> (B), Akt inhibitor VIII (C), mTORC1 inhibitor Rapamycin (D) and MEK inhibitor PD98059 were presented. Data of three independent replicates are presented as the mean+/-s.e.m., n=3. (F) HEK293 cells were treated with inhibitors including EGFR inhibitor (3 μM), LY294002 (20 μM), Akt inhibitor VIII (2 μM), Rapamycin (0.5 μM) and PD98059 (18 μM) for 6 hours. Phosphorylation of Akt on serine-473 or threonine-308, phosphorylation of S6K1 on threonine-389, expression of Pin1, tubulin and GAPDH were determined by Western Blot.
    Egfr Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egfr inhibitor/product/Millipore
    Average 91 stars, based on 9 article reviews
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    egfr inhibitor - by Bioz Stars, 2020-11
    91/100 stars
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    85
    Millipore phospho egfr tyr1086
    Effect of androgen treatment on protein abundance of AR, PSA, and cell cycle regulators in LNCaP sublines, DU-145, and PC-3 cells. Protein expression of total Akt, Akt1, Akt2, phospho-Akt Ser473, phospho-Akt Thr308, p42/44 MAPK, phospho-p42/44 MAPK Thr202/Tyr204, <t>EGFR,</t> and different phosphorylation site of tyrosine (Tyr1173, Tyr1148, <t>Tyr1086,</t> Tyr1069, Tyr1045, and Tyr845) in LNCaP 104-S, 104-R1, and 104-R2 cells treated with 0, 0.1, or 10 nM R1881 for 96 hours were assayed by Western blotting. Same GAPDH, α-tubulin, and β-actin as shown in Figure 3 were used as loading control. These proteins were also examined in PC-3 and DU-145 cells in the absence of androgen. Experiments were repeated three times. Numbers represent quantification of bands of individual protein quantified by ImageJ.
    Phospho Egfr Tyr1086, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Millipore egfr kinase activity inhibitor ag1478
    GLN's protective signaling is inhibited by the <t>EGFR</t> kinase activity inhibitor <t>AG1478.</t> A : viability of IEC-6 cells treated for 1 h with 0, 5, 10, or 20 μM AG1478 and incubated for 24 h at 37°C. CT, control. Values are means ± SE; n = 3. B : MTS assay of viability of IEC-6 cells treated as described in A . One plate was allowed to recover at 37°C for 24 h, and the other was subjected to lethal HS at 44°C for 50 min following 24 h of recovery at 37°C. All groups were normalized to their non-HS controls to account for differences in cell growth. Values are means ± SE; assays were carried out in triplicates, and experiments were performed 3 times. C : Western blot of poly(ADP-ribose) polymerase (PARP)-1 and cleaved PARP in IEC-6 cells treated with 0 or 10 mM GLN with or without 1 h of pretreatment with AG1478 (AG, 20 μM) under basal and stressed (43°C) conditions; β-actin was monitored to normalize total blotted protein. Densitometric analysis shows expression of cleaved PARP. Values are means ± SE; n = 3. D : Western blots of procaspase-3, cleaved caspase-3, and β-actin in cells treated as described in C . Densitometric analysis shows cleaved caspase-3 levels. Values are means ± SE; n = 3.
    Egfr Kinase Activity Inhibitor Ag1478, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore anti phospho egfr
    Temporal mapping of the <t>EGFR</t> cascade using site-specific inhibitor molecules. The four bar graphs represent results for the following: (a) EGF stimulation, no inhibitor, and EGF stimulation in the presence of (b) neutralizing EGFR Ab, (c) GW5074—an inhibitor of Raf kinase activity, and (d) U0126—a highly selective inhibitor of <t>MEK1/2.</t> For each inhibitor, the arrow indicates the point of pathway inhibition. Each graph contains the time course (0–20 minutes) of expression profiles for each of the four proteins analyzed. Each bar value represents the average of three replicates.
    Anti Phospho Egfr, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Inhibition of EGFR-PI3K-Akt pathway attenuates WST-8 reduction. (A~E) Increasing amount of inhibitors was used treat HEK293 cells for 6 hours. Absorbance of WST-8 formazan was recorded after incubation for 1 hour. Data of EGFR inhibitor (A), PI3K inhibitor LY294002 (B), Akt inhibitor VIII (C), mTORC1 inhibitor Rapamycin (D) and MEK inhibitor PD98059 were presented. Data of three independent replicates are presented as the mean+/-s.e.m., n=3. (F) HEK293 cells were treated with inhibitors including EGFR inhibitor (3 μM), LY294002 (20 μM), Akt inhibitor VIII (2 μM), Rapamycin (0.5 μM) and PD98059 (18 μM) for 6 hours. Phosphorylation of Akt on serine-473 or threonine-308, phosphorylation of S6K1 on threonine-389, expression of Pin1, tubulin and GAPDH were determined by Western Blot.

    Journal: International Journal of Biological Sciences

    Article Title: Cellular glucose metabolism is essential for the reduction of cell-impermeable water-soluble tetrazolium (WST) dyes

    doi: 10.7150/ijbs.25629

    Figure Lengend Snippet: Inhibition of EGFR-PI3K-Akt pathway attenuates WST-8 reduction. (A~E) Increasing amount of inhibitors was used treat HEK293 cells for 6 hours. Absorbance of WST-8 formazan was recorded after incubation for 1 hour. Data of EGFR inhibitor (A), PI3K inhibitor LY294002 (B), Akt inhibitor VIII (C), mTORC1 inhibitor Rapamycin (D) and MEK inhibitor PD98059 were presented. Data of three independent replicates are presented as the mean+/-s.e.m., n=3. (F) HEK293 cells were treated with inhibitors including EGFR inhibitor (3 μM), LY294002 (20 μM), Akt inhibitor VIII (2 μM), Rapamycin (0.5 μM) and PD98059 (18 μM) for 6 hours. Phosphorylation of Akt on serine-473 or threonine-308, phosphorylation of S6K1 on threonine-389, expression of Pin1, tubulin and GAPDH were determined by Western Blot.

    Article Snippet: GLUT inhibitor II (400035), 3-BP (376817), 3-PO (525330), EGFR inhibitor (324674), LY294002 (440202), Akt inhibitor VIII (124018), Rapamycin (553210) and PD98059 (513000) were from Millipore.

    Techniques: Inhibition, Incubation, Expressing, Western Blot

    Effect of androgen treatment on protein abundance of AR, PSA, and cell cycle regulators in LNCaP sublines, DU-145, and PC-3 cells. Protein expression of total Akt, Akt1, Akt2, phospho-Akt Ser473, phospho-Akt Thr308, p42/44 MAPK, phospho-p42/44 MAPK Thr202/Tyr204, EGFR, and different phosphorylation site of tyrosine (Tyr1173, Tyr1148, Tyr1086, Tyr1069, Tyr1045, and Tyr845) in LNCaP 104-S, 104-R1, and 104-R2 cells treated with 0, 0.1, or 10 nM R1881 for 96 hours were assayed by Western blotting. Same GAPDH, α-tubulin, and β-actin as shown in Figure 3 were used as loading control. These proteins were also examined in PC-3 and DU-145 cells in the absence of androgen. Experiments were repeated three times. Numbers represent quantification of bands of individual protein quantified by ImageJ.

    Journal: PLoS ONE

    Article Title: Difference in Protein Expression Profile and Chemotherapy Drugs Response of Different Progression Stages of LNCaP Sublines and Other Human Prostate Cancer Cells

    doi: 10.1371/journal.pone.0082625

    Figure Lengend Snippet: Effect of androgen treatment on protein abundance of AR, PSA, and cell cycle regulators in LNCaP sublines, DU-145, and PC-3 cells. Protein expression of total Akt, Akt1, Akt2, phospho-Akt Ser473, phospho-Akt Thr308, p42/44 MAPK, phospho-p42/44 MAPK Thr202/Tyr204, EGFR, and different phosphorylation site of tyrosine (Tyr1173, Tyr1148, Tyr1086, Tyr1069, Tyr1045, and Tyr845) in LNCaP 104-S, 104-R1, and 104-R2 cells treated with 0, 0.1, or 10 nM R1881 for 96 hours were assayed by Western blotting. Same GAPDH, α-tubulin, and β-actin as shown in Figure 3 were used as loading control. These proteins were also examined in PC-3 and DU-145 cells in the absence of androgen. Experiments were repeated three times. Numbers represent quantification of bands of individual protein quantified by ImageJ.

    Article Snippet: Bad, MDM2, Akt1, p42/44 MAPK, phospho-EGFR Tyr1173, phospho-EGFR Tyr1148, phospho-EGFR Tyr1086, phospho-EGFR Tyr1069, phospho-EGFR Tyr1045, and phospho-EGFR Tyr 845 were from Millipore (Billerica, MA, U.S.A.). α-tubulin was from Sigma. β-actin, α-tubulin, and GAPDH were used as loading control.

    Techniques: Expressing, Western Blot

    GLN's protective signaling is inhibited by the EGFR kinase activity inhibitor AG1478. A : viability of IEC-6 cells treated for 1 h with 0, 5, 10, or 20 μM AG1478 and incubated for 24 h at 37°C. CT, control. Values are means ± SE; n = 3. B : MTS assay of viability of IEC-6 cells treated as described in A . One plate was allowed to recover at 37°C for 24 h, and the other was subjected to lethal HS at 44°C for 50 min following 24 h of recovery at 37°C. All groups were normalized to their non-HS controls to account for differences in cell growth. Values are means ± SE; assays were carried out in triplicates, and experiments were performed 3 times. C : Western blot of poly(ADP-ribose) polymerase (PARP)-1 and cleaved PARP in IEC-6 cells treated with 0 or 10 mM GLN with or without 1 h of pretreatment with AG1478 (AG, 20 μM) under basal and stressed (43°C) conditions; β-actin was monitored to normalize total blotted protein. Densitometric analysis shows expression of cleaved PARP. Values are means ± SE; n = 3. D : Western blots of procaspase-3, cleaved caspase-3, and β-actin in cells treated as described in C . Densitometric analysis shows cleaved caspase-3 levels. Values are means ± SE; n = 3.

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Epidermal growth factor receptor expression and signaling are essential in glutamine's cytoprotective mechanism in heat-stressed intestinal epithelial-6 cells

    doi: 10.1152/ajpgi.00418.2012

    Figure Lengend Snippet: GLN's protective signaling is inhibited by the EGFR kinase activity inhibitor AG1478. A : viability of IEC-6 cells treated for 1 h with 0, 5, 10, or 20 μM AG1478 and incubated for 24 h at 37°C. CT, control. Values are means ± SE; n = 3. B : MTS assay of viability of IEC-6 cells treated as described in A . One plate was allowed to recover at 37°C for 24 h, and the other was subjected to lethal HS at 44°C for 50 min following 24 h of recovery at 37°C. All groups were normalized to their non-HS controls to account for differences in cell growth. Values are means ± SE; assays were carried out in triplicates, and experiments were performed 3 times. C : Western blot of poly(ADP-ribose) polymerase (PARP)-1 and cleaved PARP in IEC-6 cells treated with 0 or 10 mM GLN with or without 1 h of pretreatment with AG1478 (AG, 20 μM) under basal and stressed (43°C) conditions; β-actin was monitored to normalize total blotted protein. Densitometric analysis shows expression of cleaved PARP. Values are means ± SE; n = 3. D : Western blots of procaspase-3, cleaved caspase-3, and β-actin in cells treated as described in C . Densitometric analysis shows cleaved caspase-3 levels. Values are means ± SE; n = 3.

    Article Snippet: Cells were seeded in 10-cm dishes and allowed to grow for 3 days in full medium; then the medium was replaced with GLN-free serum containing DMEM for 24 h. Cells were then treated with and without 10 mM GLN for 15 min with or without 1 h of pretreatment with the EGFR kinase activity inhibitor AG1478 (20 μM), the ERK1/2 kinase inhibitor PD98059 (50 μM; Calbiochem, Philadelphia, PA), the p38MAPK inhibitor SB203580 (10 μM), or the PI3-K inhibitor (25 μM) and subjected to HS.

    Techniques: Activity Assay, Incubation, MTS Assay, Western Blot, Expressing

    ERK1/2 activation is involved in GLN's protective mechanism and attenuates after inhibition of EGFR tyrosine kinase activity. A : Western blot showing inhibitory effect of PD98059 on Thr 202 /Tyr 204 -phosphorylated [T(P) 202 /Y(P) 204 ] and total ERK1/2 levels in IEC-6 cells treated with the ERK1/2 kinase inhibitor PD98059 (50 μM) for 1 h or DMEM without GLN under basal conditions. B : representative Western blots from 3 independent experiments showing procaspase-3 and cleaved caspase-3 levels in IEC-6 cells treated with 0 or 10 mM GLN with or without 1 h of pretreatment with PD98059 (50 μM) under basal and stressed (43°C) conditions and densitometric analysis of cleaved caspase-3. Values are means ± SE; n = 3. C : Western blots showing PARP and cleaved PARP levels in IEC-6 cells treated as described in B . Blots represent results from 2 experiments. D : Western blots of Thr 202 /Tyr 204 -phosphorylated and total ERK1/2 levels under basal and heat-stressed (43°C) conditions without recovery in cells treated as described in B , but with addition of the inhibitor AG1478 (20 μM). ERK1/2 activation is shown as mean fold change relative to total ERK1/2; results were normalized to 0 mM GLN. Values are means ± SE; n = 3.

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Epidermal growth factor receptor expression and signaling are essential in glutamine's cytoprotective mechanism in heat-stressed intestinal epithelial-6 cells

    doi: 10.1152/ajpgi.00418.2012

    Figure Lengend Snippet: ERK1/2 activation is involved in GLN's protective mechanism and attenuates after inhibition of EGFR tyrosine kinase activity. A : Western blot showing inhibitory effect of PD98059 on Thr 202 /Tyr 204 -phosphorylated [T(P) 202 /Y(P) 204 ] and total ERK1/2 levels in IEC-6 cells treated with the ERK1/2 kinase inhibitor PD98059 (50 μM) for 1 h or DMEM without GLN under basal conditions. B : representative Western blots from 3 independent experiments showing procaspase-3 and cleaved caspase-3 levels in IEC-6 cells treated with 0 or 10 mM GLN with or without 1 h of pretreatment with PD98059 (50 μM) under basal and stressed (43°C) conditions and densitometric analysis of cleaved caspase-3. Values are means ± SE; n = 3. C : Western blots showing PARP and cleaved PARP levels in IEC-6 cells treated as described in B . Blots represent results from 2 experiments. D : Western blots of Thr 202 /Tyr 204 -phosphorylated and total ERK1/2 levels under basal and heat-stressed (43°C) conditions without recovery in cells treated as described in B , but with addition of the inhibitor AG1478 (20 μM). ERK1/2 activation is shown as mean fold change relative to total ERK1/2; results were normalized to 0 mM GLN. Values are means ± SE; n = 3.

    Article Snippet: Cells were seeded in 10-cm dishes and allowed to grow for 3 days in full medium; then the medium was replaced with GLN-free serum containing DMEM for 24 h. Cells were then treated with and without 10 mM GLN for 15 min with or without 1 h of pretreatment with the EGFR kinase activity inhibitor AG1478 (20 μM), the ERK1/2 kinase inhibitor PD98059 (50 μM; Calbiochem, Philadelphia, PA), the p38MAPK inhibitor SB203580 (10 μM), or the PI3-K inhibitor (25 μM) and subjected to HS.

    Techniques: Activation Assay, Inhibition, Activity Assay, Western Blot

    Temporal mapping of the EGFR cascade using site-specific inhibitor molecules. The four bar graphs represent results for the following: (a) EGF stimulation, no inhibitor, and EGF stimulation in the presence of (b) neutralizing EGFR Ab, (c) GW5074—an inhibitor of Raf kinase activity, and (d) U0126—a highly selective inhibitor of MEK1/2. For each inhibitor, the arrow indicates the point of pathway inhibition. Each graph contains the time course (0–20 minutes) of expression profiles for each of the four proteins analyzed. Each bar value represents the average of three replicates.

    Journal: International Journal of Proteomics

    Article Title: Rapid Screening of the Epidermal Growth Factor Receptor Phosphosignaling Pathway via Microplate-Based Dot Blot Assays

    doi: 10.1155/2012/473843

    Figure Lengend Snippet: Temporal mapping of the EGFR cascade using site-specific inhibitor molecules. The four bar graphs represent results for the following: (a) EGF stimulation, no inhibitor, and EGF stimulation in the presence of (b) neutralizing EGFR Ab, (c) GW5074—an inhibitor of Raf kinase activity, and (d) U0126—a highly selective inhibitor of MEK1/2. For each inhibitor, the arrow indicates the point of pathway inhibition. Each graph contains the time course (0–20 minutes) of expression profiles for each of the four proteins analyzed. Each bar value represents the average of three replicates.

    Article Snippet: As part of the initial screening process, we reviewed the certificate of analysis for each of the four antibodies: anti-phospho-EGFR (TYR1069, clone 9H2), anti-phospho-Mek1/2 (SER218/SER222, clone E237), anti-phospho-Erk 1/2 (THR202/TYR204, clone 12D4), and anti-GAPDH (clone 6C5) employed in this study (all Abs are from EMD Millipore).

    Techniques: Activity Assay, Inhibition, Expressing