anti egfr  (Millipore)


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  • 99
    Name:
    Anti EGFR antibody
    Description:
    The gene epidermal growth factor receptor EGFR is mapped to human chromosome 7p12 It belongs to receptor tyrosine kinase family The protein is mainly localized in the plasma membrane
    Catalog Number:
    hpa001200
    Price:
    None
    Applications:
    Anti-EGFR antibody produced in rabbit is suitable for use in epitope mapping to study the antibody cross-reactivity with a multi-target fragment library.Anti-EGFR antibody produced in rabbit, a Prestige Antibody, is developed and validated by the Human Protein Atlas (HPA) project (www.proteinatlas.org). Each antibody is tested by immunohistochemistry against hundreds of normal and disease tissues. These images can be viewed on the Human Protein Atlas (HPA) site by clicking on the Image Gallery link. The antibodies are also tested using immunofluorescence and western blotting. To view these protocols and other useful information about Prestige Antibodies and the HPA, visit sigma.com/prestige.
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    Structured Review

    Millipore anti egfr
    Anti EGFR antibody
    The gene epidermal growth factor receptor EGFR is mapped to human chromosome 7p12 It belongs to receptor tyrosine kinase family The protein is mainly localized in the plasma membrane
    https://www.bioz.com/result/anti egfr/product/Millipore
    Average 99 stars, based on 30 article reviews
    Price from $9.99 to $1999.99
    anti egfr - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "Vemurafenib Inhibits Active PTK6 in PTEN-null Prostate Tumor Cells"

    Article Title: Vemurafenib Inhibits Active PTK6 in PTEN-null Prostate Tumor Cells

    Journal: Molecular cancer therapeutics

    doi: 10.1158/1535-7163.MCT-18-0862

    Vemurafenib inhibits activation and signaling downstream of PTK6. (A) PC3 cells stably expressing Palm-PTK6-YF were treated with vemurafenib or DMSO vehicle and incubated for the times described. Changes in phosphorylation of PTK6, FAK, and BCAR1 were monitored by immunoblotting. Activation of PTK6 is monitored by phosphorylation of Y342. (B) PC3 Vector and PC3 Palm-PTK6-YF cells were treated with vemurafenib and total cell lysates were prepared. Changes in protein levels and phosphorylation were monitored by immunoblotting. Each lane represents one plate of cells (independent experimental replicate). Quantitation of immunoblot data is presented for DMSO and vemurafenib treated Palm-PTK6-YF (Palm-YF) expressing cells in the lower panel. Relative changes in phosphorylation of PTK6 targets were normalized to total protein levels. Residual signal following vemurafenib treatment is displayed ± SEM. (C) siRNA mediated knockdown of BRAF does not inhibit PTK6 activity. Cells were transiently transfected with scrambled or BRAF targeting siRNA (SCRsi and BRAFsi) and harvested at 72 hours. Activating phosphorylation of PTK6 (PY342) and its direct substrates FAK and EGFR are not reduced following knockdown of wild type BRAF. (D) PC3 cells subjected to stable PTK6 knockdown by two shRNA targeting vectors (sh49 and sh52) or scrambled shRNA control (shSCR) were incubated with 1 μM vemurafenib or DMSO vehicle. Similar reductions in downstream signaling are detected in PC3 cells by knockdown of PTK6 (DMSO treated cells) or vemurafenib treatment (shSCR lane). Changes in protein levels and phosphorylation were monitored by immunoblotting.
    Figure Legend Snippet: Vemurafenib inhibits activation and signaling downstream of PTK6. (A) PC3 cells stably expressing Palm-PTK6-YF were treated with vemurafenib or DMSO vehicle and incubated for the times described. Changes in phosphorylation of PTK6, FAK, and BCAR1 were monitored by immunoblotting. Activation of PTK6 is monitored by phosphorylation of Y342. (B) PC3 Vector and PC3 Palm-PTK6-YF cells were treated with vemurafenib and total cell lysates were prepared. Changes in protein levels and phosphorylation were monitored by immunoblotting. Each lane represents one plate of cells (independent experimental replicate). Quantitation of immunoblot data is presented for DMSO and vemurafenib treated Palm-PTK6-YF (Palm-YF) expressing cells in the lower panel. Relative changes in phosphorylation of PTK6 targets were normalized to total protein levels. Residual signal following vemurafenib treatment is displayed ± SEM. (C) siRNA mediated knockdown of BRAF does not inhibit PTK6 activity. Cells were transiently transfected with scrambled or BRAF targeting siRNA (SCRsi and BRAFsi) and harvested at 72 hours. Activating phosphorylation of PTK6 (PY342) and its direct substrates FAK and EGFR are not reduced following knockdown of wild type BRAF. (D) PC3 cells subjected to stable PTK6 knockdown by two shRNA targeting vectors (sh49 and sh52) or scrambled shRNA control (shSCR) were incubated with 1 μM vemurafenib or DMSO vehicle. Similar reductions in downstream signaling are detected in PC3 cells by knockdown of PTK6 (DMSO treated cells) or vemurafenib treatment (shSCR lane). Changes in protein levels and phosphorylation were monitored by immunoblotting.

    Techniques Used: Activation Assay, Stable Transfection, Expressing, Incubation, Plasmid Preparation, Quantitation Assay, Activity Assay, Transfection, shRNA

    2) Product Images from "Differential expression of folate receptor 1 in medulloblastoma and the correlation with clinicopathological characters and target therapeutic potential"

    Article Title: Differential expression of folate receptor 1 in medulloblastoma and the correlation with clinicopathological characters and target therapeutic potential

    Journal: Oncotarget

    doi: 10.18632/oncotarget.15480

    Molecular mechanisms of Folr1-Ara-C functions ( A ) Targeted effects of Folr1-Ara-C on the inhibition of EGFR, pEGFR, Erk, pErk and pStat3 in Daoy and MB-wyx cells after 8 h incubation. Lysates were analyzed for EGFR/pEGFR, pErk/Erk and pStat3/Stat3 antibodies using immunoblotting. β-actin was used as the protein loading control. ( B ) Immunoblotting analysis of MMP2, MMP9, Caspase-3/-9, Bcl-2 and p53 in Daoy and MB-wyx cells incubated with indicated agents, respectively. β-actin was used as the protein loading control.
    Figure Legend Snippet: Molecular mechanisms of Folr1-Ara-C functions ( A ) Targeted effects of Folr1-Ara-C on the inhibition of EGFR, pEGFR, Erk, pErk and pStat3 in Daoy and MB-wyx cells after 8 h incubation. Lysates were analyzed for EGFR/pEGFR, pErk/Erk and pStat3/Stat3 antibodies using immunoblotting. β-actin was used as the protein loading control. ( B ) Immunoblotting analysis of MMP2, MMP9, Caspase-3/-9, Bcl-2 and p53 in Daoy and MB-wyx cells incubated with indicated agents, respectively. β-actin was used as the protein loading control.

    Techniques Used: Acetylene Reduction Assay, Inhibition, Incubation

    3) Product Images from "Increased sugar uptake promotes oncogenesis via EPAC/RAP1 and O-GlcNAc pathways"

    Article Title: Increased sugar uptake promotes oncogenesis via EPAC/RAP1 and O-GlcNAc pathways

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI63146

    Reciprocal interactions between increased glucose uptake/metabolism and other signaling pathways. Extracellular glucose is taken up by glucose transporters, including GLUT3, as well as by diffusion and is metabolized by hexokinase (HK) and glucose-6-phosphate isomerase (GPI) to enter different metabolic pathways. The glycolytic pathway includes subsequent steps mediated by PFK, ALDO, and GAPDH; LDH also supports the glycolytic pathway by production of the GAPDH coenzyme NAD+. These enzymes were all upregulated in T4-2 cells, leading to loss of integration of form and function. The sAC-EPAC-RAP1 pathway regulates β1 integrin positively, most likely via a direct link between ATP production in the glycolytic pathway and cAMP generation by sAC, which is mediated by tumor-specific PKM2-sAC association. HBP (dashed outline) is rate-limited by GFPT, which is also upregulated via activation of oncogenic signaling. Downstream O-GlcNAcylation of target proteins mediated by OGT regulates β1 integrin, EGFR, and GLUT3 expression. Inhibition of any of the key metabolic enzymes or the key signaling molecules results in suppression of all the others, reestablishment of the polarized acinar structure, and growth arrest. See Results and Discussion for details. Fonts distinguish metabolites (italic), proteins (bold), and genes (bold and italic). Colored text highlights proteins upregulated in T4-2 cells (orange), energy carrier molecules (green), and chemical reagents that do (red) or do not (purple) induce phenotypic reversion. 2DGP, 2DG-phosphate; G6P, glucose-6-phosphate; F1,6BP, fructiose-1,6-bisphosphate; GAD3P, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; 1,3BPG, 1,3-bisphosphoglycerate; GlcNAc, N-acetylglucosamine.
    Figure Legend Snippet: Reciprocal interactions between increased glucose uptake/metabolism and other signaling pathways. Extracellular glucose is taken up by glucose transporters, including GLUT3, as well as by diffusion and is metabolized by hexokinase (HK) and glucose-6-phosphate isomerase (GPI) to enter different metabolic pathways. The glycolytic pathway includes subsequent steps mediated by PFK, ALDO, and GAPDH; LDH also supports the glycolytic pathway by production of the GAPDH coenzyme NAD+. These enzymes were all upregulated in T4-2 cells, leading to loss of integration of form and function. The sAC-EPAC-RAP1 pathway regulates β1 integrin positively, most likely via a direct link between ATP production in the glycolytic pathway and cAMP generation by sAC, which is mediated by tumor-specific PKM2-sAC association. HBP (dashed outline) is rate-limited by GFPT, which is also upregulated via activation of oncogenic signaling. Downstream O-GlcNAcylation of target proteins mediated by OGT regulates β1 integrin, EGFR, and GLUT3 expression. Inhibition of any of the key metabolic enzymes or the key signaling molecules results in suppression of all the others, reestablishment of the polarized acinar structure, and growth arrest. See Results and Discussion for details. Fonts distinguish metabolites (italic), proteins (bold), and genes (bold and italic). Colored text highlights proteins upregulated in T4-2 cells (orange), energy carrier molecules (green), and chemical reagents that do (red) or do not (purple) induce phenotypic reversion. 2DGP, 2DG-phosphate; G6P, glucose-6-phosphate; F1,6BP, fructiose-1,6-bisphosphate; GAD3P, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; 1,3BPG, 1,3-bisphosphoglycerate; GlcNAc, N-acetylglucosamine.

    Techniques Used: Diffusion-based Assay, Activation Assay, Expressing, Inhibition

    4) Product Images from "HCMV Activates the IL-6-JAK-STAT3 Axis in HepG2 Cells and Primary Human Hepatocytes"

    Article Title: HCMV Activates the IL-6-JAK-STAT3 Axis in HepG2 Cells and Primary Human Hepatocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0059591

    HCMV induces IL-6-mediated activation of the JAK-STAT3 axis in HepG2 cells and PHH. (A) Time course of STAT3 activation in HepG2 cells and PHH infected with HCMV. HepG2 cells (6×10 6 cells) were left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 0.5). PHH (2×10 6 cells) were left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 1). STAT3 activation was measured by Western blotting as described in the Materials and Methods section. Unphosphorylated STAT3 and beta-actin were used as controls, and ganciclovir was used at a concentration of 5 microg/ml. (B) Time course of JAK1/JAK2 activation in HepG2 cells and PHH infected with HCMV. HepG2 cells (6×10 6 cells) were left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 0.5). PHH (2×10 6 cells) were left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 1). JAK1/JAK2 activation was measured by Western blotting, and beta-actin was used as an internal control. The histogram shows JAK activation at 2 hours post-infection as quantified using Image J 1.40 software. (C) STAT3 activation is mediated by the IL-6-JAK pathway in HepG2 cells and PHH infected with HCMV. HepG2 cells (6×10 6 cells) and PHH (2×10 6 cells) were left uninfected or infected with HCMV (MOI = 0.5) in the presence or absence of a JAK inhibitor (1 micromol/l), a STAT3 inhibitor (10 micromol/l), a neutralizing anti-IL-6R mAb (10 microg/ml), and a neutralizing anti-EGFR mAb (20 microg/ml). Cells were left uninfected or incubated with the recombinant HCMV glycoprotein gB (10 microg/ml) for 2 hours. STAT3 activation was measured by Western blotting at day 1 post-infection in PHH incubated with JAK inhibitor, STAT3 inhibitor, anti-IL-6R mAb, and in HepG2 cells incubated with JAK and STAT3 inhibitors. STAT3 activation was measured at 2 hours post-infection in HepG2 cells incubated with anti-IL-6R mAb and anti-EGFR mAb. beta-actin was used as an internal control. The histogram shows STAT3 activation as quantified using Image J 1.40 software. (D) STAT3 activation is mediated primarily by HCMV in HepG2 cells and PHH . HepG2 cells (6×10 6 cells) and PHH (2×10 6 cells) were left uninfected or infected with HCMV or UV-inactivated HCMV (AD169, MOI = 1). The activation of STAT3 and JAK2 was measured by western blot at day 3 post-infection. beta-actin was used as a control for equal loading. The histogram shows STAT3 and JAK2 activation as quantified using Image J 1.40 software. Results of western-blots are representative of two independent experiments; histograms represent means (± SD) of two independent experiments. Ab: Antibody; EGFR: Epidermal growth factor receptor; GCV: ganciclovir.
    Figure Legend Snippet: HCMV induces IL-6-mediated activation of the JAK-STAT3 axis in HepG2 cells and PHH. (A) Time course of STAT3 activation in HepG2 cells and PHH infected with HCMV. HepG2 cells (6×10 6 cells) were left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 0.5). PHH (2×10 6 cells) were left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 1). STAT3 activation was measured by Western blotting as described in the Materials and Methods section. Unphosphorylated STAT3 and beta-actin were used as controls, and ganciclovir was used at a concentration of 5 microg/ml. (B) Time course of JAK1/JAK2 activation in HepG2 cells and PHH infected with HCMV. HepG2 cells (6×10 6 cells) were left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 0.5). PHH (2×10 6 cells) were left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 1). JAK1/JAK2 activation was measured by Western blotting, and beta-actin was used as an internal control. The histogram shows JAK activation at 2 hours post-infection as quantified using Image J 1.40 software. (C) STAT3 activation is mediated by the IL-6-JAK pathway in HepG2 cells and PHH infected with HCMV. HepG2 cells (6×10 6 cells) and PHH (2×10 6 cells) were left uninfected or infected with HCMV (MOI = 0.5) in the presence or absence of a JAK inhibitor (1 micromol/l), a STAT3 inhibitor (10 micromol/l), a neutralizing anti-IL-6R mAb (10 microg/ml), and a neutralizing anti-EGFR mAb (20 microg/ml). Cells were left uninfected or incubated with the recombinant HCMV glycoprotein gB (10 microg/ml) for 2 hours. STAT3 activation was measured by Western blotting at day 1 post-infection in PHH incubated with JAK inhibitor, STAT3 inhibitor, anti-IL-6R mAb, and in HepG2 cells incubated with JAK and STAT3 inhibitors. STAT3 activation was measured at 2 hours post-infection in HepG2 cells incubated with anti-IL-6R mAb and anti-EGFR mAb. beta-actin was used as an internal control. The histogram shows STAT3 activation as quantified using Image J 1.40 software. (D) STAT3 activation is mediated primarily by HCMV in HepG2 cells and PHH . HepG2 cells (6×10 6 cells) and PHH (2×10 6 cells) were left uninfected or infected with HCMV or UV-inactivated HCMV (AD169, MOI = 1). The activation of STAT3 and JAK2 was measured by western blot at day 3 post-infection. beta-actin was used as a control for equal loading. The histogram shows STAT3 and JAK2 activation as quantified using Image J 1.40 software. Results of western-blots are representative of two independent experiments; histograms represent means (± SD) of two independent experiments. Ab: Antibody; EGFR: Epidermal growth factor receptor; GCV: ganciclovir.

    Techniques Used: Activation Assay, Infection, Western Blot, Concentration Assay, Software, Incubation, Recombinant

    5) Product Images from "The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells"

    Article Title: The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells

    Journal: Molecular Cancer

    doi: 10.1186/s12943-018-0784-2

    A proposed pathway of the NEDD4-mediated EGFR-dependent cell migration. Activated EGFR signaling elevates cytoplasmic calcium level and subsequently activates NEDD4. The activated NEDD4 is recruited to the EGFR endosomal complex and the secretary lysosomal vesicles, where NEDD4 interacts with and ubiquitinates the ESCRT complex to facilitate the engulfment of EGFR into MVB and the secretion of lysosmal cathepsin B to extracellular matrix. The secreted lysosomal cathepsin B hydrolyzes cell matrix/junction proteins and promotes cell migration
    Figure Legend Snippet: A proposed pathway of the NEDD4-mediated EGFR-dependent cell migration. Activated EGFR signaling elevates cytoplasmic calcium level and subsequently activates NEDD4. The activated NEDD4 is recruited to the EGFR endosomal complex and the secretary lysosomal vesicles, where NEDD4 interacts with and ubiquitinates the ESCRT complex to facilitate the engulfment of EGFR into MVB and the secretion of lysosmal cathepsin B to extracellular matrix. The secreted lysosomal cathepsin B hydrolyzes cell matrix/junction proteins and promotes cell migration

    Techniques Used: Migration

    NEDD4 mediates EGFR-dependent lung cancer cell migration. a , Wound healing assay of A549 cell migration. Left top panel, the knockdown of NEDD4 by shNEDD4 (lane 2) and recovery of NEDD4 upon re-introducing NEDD4 cDNA in the knockdown cells (lane 3); NEDD4-HM, high molecular weight NEDD4; NEDD4-LM, low molecular weight NEDD4. Left bottom panel, the protein level of EGFR in the lung cancer cell lines A549 and H1650 shown by immunoblotting with the cell lysates. Middle panel, photo images of the cell migration. Right panel, quantification of the EGF-stimulated cell migration area occupied after 24 h from the data of three independent experiments using the imaging software Image J (NIH). The non-EGF-treated cell migration area was subtracted by the EGF-treated cell migration area to obtain the EGF-stimulated cell migration area. b , Transwell assay of A549 cell migration. Note that the small lightly-stained round dots are pores of the transwell plates (sh NEDD4 panels). c , Wound healing assay of H1650 cells
    Figure Legend Snippet: NEDD4 mediates EGFR-dependent lung cancer cell migration. a , Wound healing assay of A549 cell migration. Left top panel, the knockdown of NEDD4 by shNEDD4 (lane 2) and recovery of NEDD4 upon re-introducing NEDD4 cDNA in the knockdown cells (lane 3); NEDD4-HM, high molecular weight NEDD4; NEDD4-LM, low molecular weight NEDD4. Left bottom panel, the protein level of EGFR in the lung cancer cell lines A549 and H1650 shown by immunoblotting with the cell lysates. Middle panel, photo images of the cell migration. Right panel, quantification of the EGF-stimulated cell migration area occupied after 24 h from the data of three independent experiments using the imaging software Image J (NIH). The non-EGF-treated cell migration area was subtracted by the EGF-treated cell migration area to obtain the EGF-stimulated cell migration area. b , Transwell assay of A549 cell migration. Note that the small lightly-stained round dots are pores of the transwell plates (sh NEDD4 panels). c , Wound healing assay of H1650 cells

    Techniques Used: Migration, Wound Healing Assay, Molecular Weight, Imaging, Software, Transwell Assay, Staining

    NEDD4 is associated with activated EGFR. a , Co-immunoprecipitation of NEDD4 with activated EGFR in lung cancer cells. Lung cancer A549 or H358 cells were serum-starved for 12 h followed by stimulation with EGF (50 ng/ml) for indicated times. EGFR was immunoprecipitated with anti-EGFR (Mab528) and detected by immunoblotting with anti-EGFR (1005) (top panels). Co-immunoprecipitated NEDD4 was detected by immunoblotting with an anti-NEDD4 (second top panels). The level of EGFR and NEDD4 in the cell lysates was also detected by immunoblotting (middle and second bottom panels). Notice that EGFR in A549 and H358 cells has an EGF-induced degradation. b , Internalized EGFR is co-localized with NEDD4. A549 cells were serum-starved for 12 h followed by stimulation with EGF (50 ng/ml) for 0 or 60 min. The cells were immuno-stained with anti-EGFR (1005) (red) and anti-NEDD4 (green). Bar, 20 μM. c , Co-expression of NEDD4 with EGFR in lung adenocarcinoma tissue. The tissue microarray containing 63 lung adenocarcinoma section samples was immunohistochemically stained with anti-EGFR or anti-NEDD4
    Figure Legend Snippet: NEDD4 is associated with activated EGFR. a , Co-immunoprecipitation of NEDD4 with activated EGFR in lung cancer cells. Lung cancer A549 or H358 cells were serum-starved for 12 h followed by stimulation with EGF (50 ng/ml) for indicated times. EGFR was immunoprecipitated with anti-EGFR (Mab528) and detected by immunoblotting with anti-EGFR (1005) (top panels). Co-immunoprecipitated NEDD4 was detected by immunoblotting with an anti-NEDD4 (second top panels). The level of EGFR and NEDD4 in the cell lysates was also detected by immunoblotting (middle and second bottom panels). Notice that EGFR in A549 and H358 cells has an EGF-induced degradation. b , Internalized EGFR is co-localized with NEDD4. A549 cells were serum-starved for 12 h followed by stimulation with EGF (50 ng/ml) for 0 or 60 min. The cells were immuno-stained with anti-EGFR (1005) (red) and anti-NEDD4 (green). Bar, 20 μM. c , Co-expression of NEDD4 with EGFR in lung adenocarcinoma tissue. The tissue microarray containing 63 lung adenocarcinoma section samples was immunohistochemically stained with anti-EGFR or anti-NEDD4

    Techniques Used: Immunoprecipitation, Staining, Expressing, Microarray

    6) Product Images from "Hepatitis C Virus Induces Epidermal Growth Factor Receptor Activation via CD81 Binding for Viral Internalization and Entry"

    Article Title: Hepatitis C Virus Induces Epidermal Growth Factor Receptor Activation via CD81 Binding for Viral Internalization and Entry

    Journal: Journal of Virology

    doi: 10.1128/JVI.00750-12

    CD81 cross-linking induces CD81-EGFR colocalization and internalization. (A) Huh-7.5 cells were incubated with FITC-labeled control or anti-CD81 antibodies for 1 h at 4°C or shifted to 37°C for another hour. Cells were fixed and stained
    Figure Legend Snippet: CD81 cross-linking induces CD81-EGFR colocalization and internalization. (A) Huh-7.5 cells were incubated with FITC-labeled control or anti-CD81 antibodies for 1 h at 4°C or shifted to 37°C for another hour. Cells were fixed and stained

    Techniques Used: Incubation, Labeling, Staining

    7) Product Images from "High glucose levels promote the proliferation of breast cancer cells through GTPases"

    Article Title: High glucose levels promote the proliferation of breast cancer cells through GTPases

    Journal: Breast Cancer : Targets and Therapy

    doi: 10.2147/BCTT.S135665

    High glucose regulates EGFR activity through GTPase. Notes: ( A ) Serum-deprived MDAMB231 cells expressing scrambled (lanes 1 and 2) or Rac1 RNAis (lanes 3 and 4) were cultured in low glucose condition (5 mM) for 12 hours and then exposed to high glucose (25 mM). After 6 hours, these cells were harvested for the measurement of EGFR phosphorylation, total EGFR levels and EGFR ubiquitination. The Western blot analysis of EGFR phosphorylation was quantified and presented by the plot at the bottom. ( B ) Serum-deprived MDAMB231 cells expressing scrambled (lanes 1 and 2) or Cdc42 RNAis (lanes 3 and 4) were cultured in low glucose condition (5 mM) for 12 hours and then exposed to high glucose (25 mM) with serum (5% FBS). After 6 hours, these cells were harvested for measurement of EGFR phosphorylation, total EGFR levels, the ubiquitination of EGFR, c-cbl phosphorylation, and total c-cbl levels. The Western blot analysis of EGFR phosphorylation and EGFR ubiquitination were quantified and presented by the plots at the bottom. ( C ) Serum-deprived MDAMB231 cells expressing HA-tagged Rac1 (G12V) (lane 2) were cultured in low glucose condition (5 mM) for 12 hours and then were harvested for measurement of EGFR phosphorylation and total EGFR levels. The Western blot analysis of EGFR phosphorylation was quantified and presented by the plot at the bottom. ( D ) Serum-deprived MDAMB231 cells expressing HA-tagged Cdc42 (F28L) (lane 2) were cultured in low glucose condition (5 mM) for 12 hours and then exposed to high glucose (25 mM, lane 3) with serum (5% FBS). After 6 hours, these cells were harvested for measurement of EGFR phosphorylation, total EGFR levels, and the ubiquitination of EGFR. The Western blot analysis of EGFR phosphorylation and EGFR ubiquitination were quantified and presented by the plots at the bottom. Abbreviations: EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; HG, high glucose; LG, low glucose; WB, western blot; WCL, whole cell lysates.
    Figure Legend Snippet: High glucose regulates EGFR activity through GTPase. Notes: ( A ) Serum-deprived MDAMB231 cells expressing scrambled (lanes 1 and 2) or Rac1 RNAis (lanes 3 and 4) were cultured in low glucose condition (5 mM) for 12 hours and then exposed to high glucose (25 mM). After 6 hours, these cells were harvested for the measurement of EGFR phosphorylation, total EGFR levels and EGFR ubiquitination. The Western blot analysis of EGFR phosphorylation was quantified and presented by the plot at the bottom. ( B ) Serum-deprived MDAMB231 cells expressing scrambled (lanes 1 and 2) or Cdc42 RNAis (lanes 3 and 4) were cultured in low glucose condition (5 mM) for 12 hours and then exposed to high glucose (25 mM) with serum (5% FBS). After 6 hours, these cells were harvested for measurement of EGFR phosphorylation, total EGFR levels, the ubiquitination of EGFR, c-cbl phosphorylation, and total c-cbl levels. The Western blot analysis of EGFR phosphorylation and EGFR ubiquitination were quantified and presented by the plots at the bottom. ( C ) Serum-deprived MDAMB231 cells expressing HA-tagged Rac1 (G12V) (lane 2) were cultured in low glucose condition (5 mM) for 12 hours and then were harvested for measurement of EGFR phosphorylation and total EGFR levels. The Western blot analysis of EGFR phosphorylation was quantified and presented by the plot at the bottom. ( D ) Serum-deprived MDAMB231 cells expressing HA-tagged Cdc42 (F28L) (lane 2) were cultured in low glucose condition (5 mM) for 12 hours and then exposed to high glucose (25 mM, lane 3) with serum (5% FBS). After 6 hours, these cells were harvested for measurement of EGFR phosphorylation, total EGFR levels, and the ubiquitination of EGFR. The Western blot analysis of EGFR phosphorylation and EGFR ubiquitination were quantified and presented by the plots at the bottom. Abbreviations: EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; HG, high glucose; LG, low glucose; WB, western blot; WCL, whole cell lysates.

    Techniques Used: Activity Assay, Expressing, Cell Culture, Western Blot

    8) Product Images from "Sentinel lymph node biopsy revisited: ultrasound-guided photoacoustic detection of micrometastases using molecularly targeted plasmonic nanosensors"

    Article Title: Sentinel lymph node biopsy revisited: ultrasound-guided photoacoustic detection of micrometastases using molecularly targeted plasmonic nanosensors

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-14-0796

    Optical hyperspectral microscopy of specific nanoparticle uptake and plasmon resonance coupling of MAPS. H E stains of metastases (dashed red outline) in mice injected with either ( a ) EGFR-targeted MAPS or ( b ) RG16-targeted AuNPs. ( c and d ) Dark-field
    Figure Legend Snippet: Optical hyperspectral microscopy of specific nanoparticle uptake and plasmon resonance coupling of MAPS. H E stains of metastases (dashed red outline) in mice injected with either ( a ) EGFR-targeted MAPS or ( b ) RG16-targeted AuNPs. ( c and d ) Dark-field

    Techniques Used: Microscopy, Mouse Assay, Injection

    9) Product Images from "Production of a Functional Factor, p40, by Lactobacillus rhamnosus GG Is Promoted by Intestinal Epithelial Cell-Secreted Extracellular Vesicles"

    Article Title: Production of a Functional Factor, p40, by Lactobacillus rhamnosus GG Is Promoted by Intestinal Epithelial Cell-Secreted Extracellular Vesicles

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00113-19

    Intestinal epithelial cell-derived components promote the protective effects of LGG on intestinal epithelial cells. YAMC-CM was prepared from 24-h cell culture. LGG was cultured in RPMI (RPMI-LGG) or YAMC-CM (YAMC-CM-LGG) for 3 h at 37°C. (A) YAMC were treated with LGG, RPMI-LGG, or YAMC-CM-LGG at 10 7 CFU/ml for 1 h. (B) HT29 cells were treated with TNF (100 ng/ml), IL-1α (10 ng/ml), and IFN-γ (100 ng/ml) for 6 h in the presence or absence of LGG, RPMI-LGG, or YAMC-CM-LGG. Cellular lysates were collected for Western blot analysis of levels of phosphorylated ERGF (P-EGFR) and total EGFR (T-EGFR) (A) and cleavage caspase-3 (B). β-Actin was used as a protein loading control. The relative density was determined by normalization of the band density of P-EGFR to that of T-EGFR and cleavage caspase-3 to that of β-actin in the same sample. The fold change (shown under P-EGFR and cleavage caspase-3 blots) was calculated by comparison of the relative density in each sample to that of P-EGFR in nontreated YAMC (A) and cleavage caspase-3 in HT29 cells treated with cytokines only (B). (C) T84 cells were treated with H 2 O 2 (20 μM) for 3 h in the presence or absence of RPMI-LGG or YAMC-CM-LGG. The cotreatment was present during H 2 O 2 treatment. Cells were fixed for immunostaining of ZO-1. Data are representative of at least 3 independent experiments.
    Figure Legend Snippet: Intestinal epithelial cell-derived components promote the protective effects of LGG on intestinal epithelial cells. YAMC-CM was prepared from 24-h cell culture. LGG was cultured in RPMI (RPMI-LGG) or YAMC-CM (YAMC-CM-LGG) for 3 h at 37°C. (A) YAMC were treated with LGG, RPMI-LGG, or YAMC-CM-LGG at 10 7 CFU/ml for 1 h. (B) HT29 cells were treated with TNF (100 ng/ml), IL-1α (10 ng/ml), and IFN-γ (100 ng/ml) for 6 h in the presence or absence of LGG, RPMI-LGG, or YAMC-CM-LGG. Cellular lysates were collected for Western blot analysis of levels of phosphorylated ERGF (P-EGFR) and total EGFR (T-EGFR) (A) and cleavage caspase-3 (B). β-Actin was used as a protein loading control. The relative density was determined by normalization of the band density of P-EGFR to that of T-EGFR and cleavage caspase-3 to that of β-actin in the same sample. The fold change (shown under P-EGFR and cleavage caspase-3 blots) was calculated by comparison of the relative density in each sample to that of P-EGFR in nontreated YAMC (A) and cleavage caspase-3 in HT29 cells treated with cytokines only (B). (C) T84 cells were treated with H 2 O 2 (20 μM) for 3 h in the presence or absence of RPMI-LGG or YAMC-CM-LGG. The cotreatment was present during H 2 O 2 treatment. Cells were fixed for immunostaining of ZO-1. Data are representative of at least 3 independent experiments.

    Techniques Used: Derivative Assay, Cell Culture, Western Blot, Immunostaining

    10) Product Images from "CD4+ T-helper Type 1 Cytokines and Trastuzumab Facilitate CD8+ T-cell Targeting of HER-2/neu-expressing Cancers"

    Article Title: CD4+ T-helper Type 1 Cytokines and Trastuzumab Facilitate CD8+ T-cell Targeting of HER-2/neu-expressing Cancers

    Journal: Cancer immunology research

    doi: 10.1158/2326-6066.CIR-14-0208

    Resistance to Th1 cytokine/trastuzumab-mediated class I restoration and HER2 369-377 -CD8 + T-cell targeting of HER2-expressing cancers by EGF/Heregulin is rescued with inhibition of EGFR and HER3 signaling (A) Effect of EGFR- and HER3-mediated signaling on class I restoration by trastuzumab and Th1 cytokines. Trastuzumab-treated HER2 high BT-474 cells were serum starved and activated with EGF, Heregulin, or both, followed by IFNγ and TNFα treatment. Harvested cells were assessed for HLA-ABC expression by flow cytometry. Results are representative of three experiments, and expressed as mean HLA-ABC MCF ± SEM. (B) Trastuzumab/IFNγ/TNFα-treated HER2 high SK-BR-3 ( left panel ) and BT-474 ( right panel ) cells with or without EGF + Heregulin activation, were subsequently treated with anti-EGFR + anti-HER3 neutralizing or IgG1 isotype control antibodies. Harvested cells were assessed for HLA-ABC expression by flow cytometry. In representative panels, filled traces represent isotype-matched control staining, and open traces represent HLA-ABC staining. Color-coded cell treatments are indicated in the legend. Adjoining results in histograms are representative of three experiments, and expressed as mean HLA-ABC MCF ± SEM; cell treatments are indicated below the histograms. (C) Following treatments indicated in (B) above, CFSE-labeled HER2 high SK-BR-3 cells were co-cultured 1:1 with HER2 369-377 -sensitized CD8 + T cells. Tumor cells were harvested, stained with 7-AAD and FITC:anti-CD8, and CSFE + 7-AAD + CD8 - cells (apoptotic) were assessed by flow cytometry. Results are representative of three experiments, and expressed as % apoptotic tumor cells±SEM ( % lysis ) in co-culture minus background. *p≤0.05, **p
    Figure Legend Snippet: Resistance to Th1 cytokine/trastuzumab-mediated class I restoration and HER2 369-377 -CD8 + T-cell targeting of HER2-expressing cancers by EGF/Heregulin is rescued with inhibition of EGFR and HER3 signaling (A) Effect of EGFR- and HER3-mediated signaling on class I restoration by trastuzumab and Th1 cytokines. Trastuzumab-treated HER2 high BT-474 cells were serum starved and activated with EGF, Heregulin, or both, followed by IFNγ and TNFα treatment. Harvested cells were assessed for HLA-ABC expression by flow cytometry. Results are representative of three experiments, and expressed as mean HLA-ABC MCF ± SEM. (B) Trastuzumab/IFNγ/TNFα-treated HER2 high SK-BR-3 ( left panel ) and BT-474 ( right panel ) cells with or without EGF + Heregulin activation, were subsequently treated with anti-EGFR + anti-HER3 neutralizing or IgG1 isotype control antibodies. Harvested cells were assessed for HLA-ABC expression by flow cytometry. In representative panels, filled traces represent isotype-matched control staining, and open traces represent HLA-ABC staining. Color-coded cell treatments are indicated in the legend. Adjoining results in histograms are representative of three experiments, and expressed as mean HLA-ABC MCF ± SEM; cell treatments are indicated below the histograms. (C) Following treatments indicated in (B) above, CFSE-labeled HER2 high SK-BR-3 cells were co-cultured 1:1 with HER2 369-377 -sensitized CD8 + T cells. Tumor cells were harvested, stained with 7-AAD and FITC:anti-CD8, and CSFE + 7-AAD + CD8 - cells (apoptotic) were assessed by flow cytometry. Results are representative of three experiments, and expressed as % apoptotic tumor cells±SEM ( % lysis ) in co-culture minus background. *p≤0.05, **p

    Techniques Used: Expressing, Inhibition, Flow Cytometry, Cytometry, Activation Assay, Staining, Labeling, Cell Culture, Lysis, Co-Culture Assay

    11) Product Images from "Sema3E-Plexin D1 signaling drives human cancer cell invasiveness and metastatic spreading in mice"

    Article Title: Sema3E-Plexin D1 signaling drives human cancer cell invasiveness and metastatic spreading in mice

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI42118

    p61-Sema3E elicits the activation of Plexin D1–associated tyrosine kinase ErbB2. ( A ) A549 tumor cells were starved for 48 hours and stimulated for 15 minutes with 7 nM p61-Sema3E or 0.2 nM Heregulin-β1 ECD (Hrg-β). ErbB2 was immunoprecipitated and analyzed by immunoblotting with anti–phospho-tyrosine (anti-pY) antibodies. The graph shows the average fold change of band intensity ± SD observed in 3 experiments (normalized to controls). ( B ) As above, serum-starved A549 cells were stimulated for 15 minutes with 7 nM p61 or 1 nM EGF. Then, EGFR or ErbB3 were immunoprecipitated using appropriate antibodies and analyzed with anti–phospho-tyrosine antibodies. ( C ) In analogy to the above experiments, ErbB2 phosphorylation in response to p61 or Hrg-β was assayed in HeLa carcinoma cells, either control or Plexin D1 depleted (verified by Western blot analysis). The graph shows the average fold change of band intensity ± SD observed in 3 experiments. ( D ) Plexin D1 (VSV-tagged) and ErbB2, transfected into HEK293 cells, coprecipitate in a specific complex, as revealed by immunoblotting. Analogous results were obtained upon transfection in COS cells (data not shown). The experiment was repeated 3 times with consistent results. ( E ) COS cells transfected with VSV-tagged Plexin D1 were preincubated with either 200 nM Lapatinib, or 250 nM PHA-665752 (PHA), or vehicle, for 3 hours. Thereafter, cells were treated for 15 minutes with 7 nM p61 or mock stimulated. Plexin D1 was immunoprecipitated and analyzed with anti–phospho-tyrosine antibodies. ( F ) Serum-starved A549 cancer cells, expressing endogenous Plexin D1 and ErbB2 receptors, were treated with mock or 7 nM p61-Sema3E for 10 minutes. Plexin D1 copurifying with ErbB2 was revealed by immunoblotting.
    Figure Legend Snippet: p61-Sema3E elicits the activation of Plexin D1–associated tyrosine kinase ErbB2. ( A ) A549 tumor cells were starved for 48 hours and stimulated for 15 minutes with 7 nM p61-Sema3E or 0.2 nM Heregulin-β1 ECD (Hrg-β). ErbB2 was immunoprecipitated and analyzed by immunoblotting with anti–phospho-tyrosine (anti-pY) antibodies. The graph shows the average fold change of band intensity ± SD observed in 3 experiments (normalized to controls). ( B ) As above, serum-starved A549 cells were stimulated for 15 minutes with 7 nM p61 or 1 nM EGF. Then, EGFR or ErbB3 were immunoprecipitated using appropriate antibodies and analyzed with anti–phospho-tyrosine antibodies. ( C ) In analogy to the above experiments, ErbB2 phosphorylation in response to p61 or Hrg-β was assayed in HeLa carcinoma cells, either control or Plexin D1 depleted (verified by Western blot analysis). The graph shows the average fold change of band intensity ± SD observed in 3 experiments. ( D ) Plexin D1 (VSV-tagged) and ErbB2, transfected into HEK293 cells, coprecipitate in a specific complex, as revealed by immunoblotting. Analogous results were obtained upon transfection in COS cells (data not shown). The experiment was repeated 3 times with consistent results. ( E ) COS cells transfected with VSV-tagged Plexin D1 were preincubated with either 200 nM Lapatinib, or 250 nM PHA-665752 (PHA), or vehicle, for 3 hours. Thereafter, cells were treated for 15 minutes with 7 nM p61 or mock stimulated. Plexin D1 was immunoprecipitated and analyzed with anti–phospho-tyrosine antibodies. ( F ) Serum-starved A549 cancer cells, expressing endogenous Plexin D1 and ErbB2 receptors, were treated with mock or 7 nM p61-Sema3E for 10 minutes. Plexin D1 copurifying with ErbB2 was revealed by immunoblotting.

    Techniques Used: Activation Assay, Immunoprecipitation, Western Blot, Transfection, Expressing

    12) Product Images from "The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells"

    Article Title: The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells

    Journal: Molecular Cancer

    doi: 10.1186/s12943-018-0784-2

    A proposed pathway of the NEDD4-mediated EGFR-dependent cell migration. Activated EGFR signaling elevates cytoplasmic calcium level and subsequently activates NEDD4. The activated NEDD4 is recruited to the EGFR endosomal complex and the secretary lysosomal vesicles, where NEDD4 interacts with and ubiquitinates the ESCRT complex to facilitate the engulfment of EGFR into MVB and the secretion of lysosmal cathepsin B to extracellular matrix. The secreted lysosomal cathepsin B hydrolyzes cell matrix/junction proteins and promotes cell migration
    Figure Legend Snippet: A proposed pathway of the NEDD4-mediated EGFR-dependent cell migration. Activated EGFR signaling elevates cytoplasmic calcium level and subsequently activates NEDD4. The activated NEDD4 is recruited to the EGFR endosomal complex and the secretary lysosomal vesicles, where NEDD4 interacts with and ubiquitinates the ESCRT complex to facilitate the engulfment of EGFR into MVB and the secretion of lysosmal cathepsin B to extracellular matrix. The secreted lysosomal cathepsin B hydrolyzes cell matrix/junction proteins and promotes cell migration

    Techniques Used: Migration

    NEDD4 mediates EGFR-dependent lung cancer cell migration. a , Wound healing assay of A549 cell migration. Left top panel, the knockdown of NEDD4 by shNEDD4 (lane 2) and recovery of NEDD4 upon re-introducing NEDD4 cDNA in the knockdown cells (lane 3); NEDD4-HM, high molecular weight NEDD4; NEDD4-LM, low molecular weight NEDD4. Left bottom panel, the protein level of EGFR in the lung cancer cell lines A549 and H1650 shown by immunoblotting with the cell lysates. Middle panel, photo images of the cell migration. Right panel, quantification of the EGF-stimulated cell migration area occupied after 24 h from the data of three independent experiments using the imaging software Image J (NIH). The non-EGF-treated cell migration area was subtracted by the EGF-treated cell migration area to obtain the EGF-stimulated cell migration area. b , Transwell assay of A549 cell migration. Note that the small lightly-stained round dots are pores of the transwell plates (sh NEDD4 panels). c , Wound healing assay of H1650 cells
    Figure Legend Snippet: NEDD4 mediates EGFR-dependent lung cancer cell migration. a , Wound healing assay of A549 cell migration. Left top panel, the knockdown of NEDD4 by shNEDD4 (lane 2) and recovery of NEDD4 upon re-introducing NEDD4 cDNA in the knockdown cells (lane 3); NEDD4-HM, high molecular weight NEDD4; NEDD4-LM, low molecular weight NEDD4. Left bottom panel, the protein level of EGFR in the lung cancer cell lines A549 and H1650 shown by immunoblotting with the cell lysates. Middle panel, photo images of the cell migration. Right panel, quantification of the EGF-stimulated cell migration area occupied after 24 h from the data of three independent experiments using the imaging software Image J (NIH). The non-EGF-treated cell migration area was subtracted by the EGF-treated cell migration area to obtain the EGF-stimulated cell migration area. b , Transwell assay of A549 cell migration. Note that the small lightly-stained round dots are pores of the transwell plates (sh NEDD4 panels). c , Wound healing assay of H1650 cells

    Techniques Used: Migration, Wound Healing Assay, Molecular Weight, Imaging, Software, Transwell Assay, Staining

    NEDD4 is associated with activated EGFR. a , Co-immunoprecipitation of NEDD4 with activated EGFR in lung cancer cells. Lung cancer A549 or H358 cells were serum-starved for 12 h followed by stimulation with EGF (50 ng/ml) for indicated times. EGFR was immunoprecipitated with anti-EGFR (Mab528) and detected by immunoblotting with anti-EGFR (1005) (top panels). Co-immunoprecipitated NEDD4 was detected by immunoblotting with an anti-NEDD4 (second top panels). The level of EGFR and NEDD4 in the cell lysates was also detected by immunoblotting (middle and second bottom panels). Notice that EGFR in A549 and H358 cells has an EGF-induced degradation. b , Internalized EGFR is co-localized with NEDD4. A549 cells were serum-starved for 12 h followed by stimulation with EGF (50 ng/ml) for 0 or 60 min. The cells were immuno-stained with anti-EGFR (1005) (red) and anti-NEDD4 (green). Bar, 20 μM. c , Co-expression of NEDD4 with EGFR in lung adenocarcinoma tissue. The tissue microarray containing 63 lung adenocarcinoma section samples was immunohistochemically stained with anti-EGFR or anti-NEDD4
    Figure Legend Snippet: NEDD4 is associated with activated EGFR. a , Co-immunoprecipitation of NEDD4 with activated EGFR in lung cancer cells. Lung cancer A549 or H358 cells were serum-starved for 12 h followed by stimulation with EGF (50 ng/ml) for indicated times. EGFR was immunoprecipitated with anti-EGFR (Mab528) and detected by immunoblotting with anti-EGFR (1005) (top panels). Co-immunoprecipitated NEDD4 was detected by immunoblotting with an anti-NEDD4 (second top panels). The level of EGFR and NEDD4 in the cell lysates was also detected by immunoblotting (middle and second bottom panels). Notice that EGFR in A549 and H358 cells has an EGF-induced degradation. b , Internalized EGFR is co-localized with NEDD4. A549 cells were serum-starved for 12 h followed by stimulation with EGF (50 ng/ml) for 0 or 60 min. The cells were immuno-stained with anti-EGFR (1005) (red) and anti-NEDD4 (green). Bar, 20 μM. c , Co-expression of NEDD4 with EGFR in lung adenocarcinoma tissue. The tissue microarray containing 63 lung adenocarcinoma section samples was immunohistochemically stained with anti-EGFR or anti-NEDD4

    Techniques Used: Immunoprecipitation, Staining, Expressing, Microarray

    13) Product Images from "The MARCH Family E3 Ubiquitin Ligase K5 Alters Monocyte Metabolism and Proliferation through Receptor Tyrosine Kinase Modulation"

    Article Title: The MARCH Family E3 Ubiquitin Ligase K5 Alters Monocyte Metabolism and Proliferation through Receptor Tyrosine Kinase Modulation

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1001331

    K5 interacts with and alters RTK localization. Equal cell numbers of the indicated THP-1 lines were fixed with paraformaldehyde (PFA) and ( A ) stained without permeabilization to determine surface expression or ( B ) permeabilized with saponin prior to staining to determine total expression of Flt-4, PDGFR-ß, Flt-3 and EGFR by flow cytometry. Data are representative of three independent experiments. ( B, Inset ) The relative ratio of surface versus total RTKs was determined for vector- and K5 WT-expressing THP-1 cells. ( C ) 293T cells were co-transfected with expression constructs for EGFR, PDGFR-ß, or Flt-4 and the indicated GST expression constructs. After two days, lysates were subjected to GST pull-down using glutathione-sepharose beads. Purified proteins and whole cell lysates (WCL) were subjected to western blot (WB) using anti-EGFR, -Flt-4 or -PDGFR-ß antibodies, followed by re-probing with anti-GST antibodies. Arrows indicate GST or GST-K5 WT and mutant specific bands. Data are representative of three independent experiments.
    Figure Legend Snippet: K5 interacts with and alters RTK localization. Equal cell numbers of the indicated THP-1 lines were fixed with paraformaldehyde (PFA) and ( A ) stained without permeabilization to determine surface expression or ( B ) permeabilized with saponin prior to staining to determine total expression of Flt-4, PDGFR-ß, Flt-3 and EGFR by flow cytometry. Data are representative of three independent experiments. ( B, Inset ) The relative ratio of surface versus total RTKs was determined for vector- and K5 WT-expressing THP-1 cells. ( C ) 293T cells were co-transfected with expression constructs for EGFR, PDGFR-ß, or Flt-4 and the indicated GST expression constructs. After two days, lysates were subjected to GST pull-down using glutathione-sepharose beads. Purified proteins and whole cell lysates (WCL) were subjected to western blot (WB) using anti-EGFR, -Flt-4 or -PDGFR-ß antibodies, followed by re-probing with anti-GST antibodies. Arrows indicate GST or GST-K5 WT and mutant specific bands. Data are representative of three independent experiments.

    Techniques Used: Staining, Expressing, Flow Cytometry, Cytometry, Plasmid Preparation, Transfection, Construct, Purification, Western Blot, Mutagenesis

    14) Product Images from "Loss of PRP4K drives anoikis resistance in part by dysregulation of epidermal growth factor receptor endosomal trafficking"

    Article Title: Loss of PRP4K drives anoikis resistance in part by dysregulation of epidermal growth factor receptor endosomal trafficking

    Journal: Oncogene

    doi: 10.1038/onc.2017.318

    PRP4K regulates EGF-dependent EGFR degradation. ( a ) HeLa shCTRL, shPRP4K-1 and shPRP4K-2 cells were serum starved overnight and treated with 50 ng/ml EGF for 30 and 90 min. Cells were fixed and analyzed by immunofluorescence confocal microscopy using an antibody against phospho-EGFR (green). Nuclei were stained with DAPI (blue). White boxes outline the cell shown at an increased magnification directly below. Scale bars, 10 microns. ( b ) HeLa shCTRL, shPRP4K-1 and shPRP4K-2 cells were serum starved overnight and treated with 50 ng/ml EGF for the indicated time period. Whole-cell lysates were prepared and subject to western blot analysis using the indicated antibodies. ( c ) Cytoplasmic phospho-EGFR (pEGFR) levels (90 min post-EGF stimulation) where quantified by immunofluorescence microscopy as a percentage of pEGFR staining at 30 min post EGF in each cell line. The points in the scatterplots represent the sum of the mean fluorescence intensity of all cytoplasmic pEGFR puncta per cell from 12 fields of view (z-stack projections of 20 confocal sections captured at 0.4 μm intervals) across three experiments, where each field of view contained 20–25 cells. Error bars=s.e.m. *** P
    Figure Legend Snippet: PRP4K regulates EGF-dependent EGFR degradation. ( a ) HeLa shCTRL, shPRP4K-1 and shPRP4K-2 cells were serum starved overnight and treated with 50 ng/ml EGF for 30 and 90 min. Cells were fixed and analyzed by immunofluorescence confocal microscopy using an antibody against phospho-EGFR (green). Nuclei were stained with DAPI (blue). White boxes outline the cell shown at an increased magnification directly below. Scale bars, 10 microns. ( b ) HeLa shCTRL, shPRP4K-1 and shPRP4K-2 cells were serum starved overnight and treated with 50 ng/ml EGF for the indicated time period. Whole-cell lysates were prepared and subject to western blot analysis using the indicated antibodies. ( c ) Cytoplasmic phospho-EGFR (pEGFR) levels (90 min post-EGF stimulation) where quantified by immunofluorescence microscopy as a percentage of pEGFR staining at 30 min post EGF in each cell line. The points in the scatterplots represent the sum of the mean fluorescence intensity of all cytoplasmic pEGFR puncta per cell from 12 fields of view (z-stack projections of 20 confocal sections captured at 0.4 μm intervals) across three experiments, where each field of view contained 20–25 cells. Error bars=s.e.m. *** P

    Techniques Used: Immunofluorescence, Confocal Microscopy, Staining, Western Blot, Microscopy, Fluorescence

    15) Product Images from "Epidermal Growth Factor Receptor (EGFR) Signaling Requires a Specific Endoplasmic Reticulum Thioredoxin for the Post-translational Control of Receptor Presentation to the Cell Surface *"

    Article Title: Epidermal Growth Factor Receptor (EGFR) Signaling Requires a Specific Endoplasmic Reticulum Thioredoxin for the Post-translational Control of Receptor Presentation to the Cell Surface *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.623207

    Recombinant AGR2 expression in MCF-10A and CHO-K1 cells results in EGFR plasma membrane delivery. A , immunocytochemistry of permeabilized MCF-10A cells with anti-cytoplasmic ( red , top panels ) or mAb 528 ( green , bottom panels ) EGFR antibodies. The cells
    Figure Legend Snippet: Recombinant AGR2 expression in MCF-10A and CHO-K1 cells results in EGFR plasma membrane delivery. A , immunocytochemistry of permeabilized MCF-10A cells with anti-cytoplasmic ( red , top panels ) or mAb 528 ( green , bottom panels ) EGFR antibodies. The cells

    Techniques Used: Recombinant, Expressing, Immunocytochemistry

    Reduced AGR2 expression decreases EGFR signaling in tyrosine kinase inhibitor-resistant cells. A and B , quantitative real-time-PCR analysis of EGR1 ( A ) and FOS ( B ) RNA in NCI-H460 cells treated with AG1478 (2 μ m ) and shAGR2. C and D , effect of
    Figure Legend Snippet: Reduced AGR2 expression decreases EGFR signaling in tyrosine kinase inhibitor-resistant cells. A and B , quantitative real-time-PCR analysis of EGR1 ( A ) and FOS ( B ) RNA in NCI-H460 cells treated with AG1478 (2 μ m ) and shAGR2. C and D , effect of

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    EGFR-mediated signaling is dependent on AGR2 expression. A , protein immunoblots for EGFR, phosphorylated EGFR ( EGFR-P ), EGR1, FOS, and β-actin ( ACTB ) of whole cell lysates derived from A431 cells propagated in serum-containing media after transduction
    Figure Legend Snippet: EGFR-mediated signaling is dependent on AGR2 expression. A , protein immunoblots for EGFR, phosphorylated EGFR ( EGFR-P ), EGR1, FOS, and β-actin ( ACTB ) of whole cell lysates derived from A431 cells propagated in serum-containing media after transduction

    Techniques Used: Expressing, Western Blot, Derivative Assay, Transduction

    AGR2 determines EGFR cell surface expression. Immunofluorescence with anti-EGFR mAb 528 ( A ) or anti-ITGB1 antibodies ( B ) to label non-permeabilized NCI-H460 and A431 cells transduced with a vector control or shAGR2. The nuclei were labeled with DAPI stain
    Figure Legend Snippet: AGR2 determines EGFR cell surface expression. Immunofluorescence with anti-EGFR mAb 528 ( A ) or anti-ITGB1 antibodies ( B ) to label non-permeabilized NCI-H460 and A431 cells transduced with a vector control or shAGR2. The nuclei were labeled with DAPI stain

    Techniques Used: Expressing, Immunofluorescence, Transduction, Plasmid Preparation, Labeling, Staining

    16) Product Images from "Epidermal Growth Factor Receptor (EGFR) Signaling Requires a Specific Endoplasmic Reticulum Thioredoxin for the Post-translational Control of Receptor Presentation to the Cell Surface *"

    Article Title: Epidermal Growth Factor Receptor (EGFR) Signaling Requires a Specific Endoplasmic Reticulum Thioredoxin for the Post-translational Control of Receptor Presentation to the Cell Surface *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.623207

    Recombinant AGR2 expression in MCF-10A and CHO-K1 cells results in EGFR plasma membrane delivery. A , immunocytochemistry of permeabilized MCF-10A cells with anti-cytoplasmic ( red , top panels ) or mAb 528 ( green , bottom panels ) EGFR antibodies. The cells
    Figure Legend Snippet: Recombinant AGR2 expression in MCF-10A and CHO-K1 cells results in EGFR plasma membrane delivery. A , immunocytochemistry of permeabilized MCF-10A cells with anti-cytoplasmic ( red , top panels ) or mAb 528 ( green , bottom panels ) EGFR antibodies. The cells

    Techniques Used: Recombinant, Expressing, Immunocytochemistry

    Reduced AGR2 expression decreases EGFR signaling in tyrosine kinase inhibitor-resistant cells. A and B , quantitative real-time-PCR analysis of EGR1 ( A ) and FOS ( B ) RNA in NCI-H460 cells treated with AG1478 (2 μ m ) and shAGR2. C and D , effect of
    Figure Legend Snippet: Reduced AGR2 expression decreases EGFR signaling in tyrosine kinase inhibitor-resistant cells. A and B , quantitative real-time-PCR analysis of EGR1 ( A ) and FOS ( B ) RNA in NCI-H460 cells treated with AG1478 (2 μ m ) and shAGR2. C and D , effect of

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    EGFR-mediated signaling is dependent on AGR2 expression. A , protein immunoblots for EGFR, phosphorylated EGFR ( EGFR-P ), EGR1, FOS, and β-actin ( ACTB ) of whole cell lysates derived from A431 cells propagated in serum-containing media after transduction
    Figure Legend Snippet: EGFR-mediated signaling is dependent on AGR2 expression. A , protein immunoblots for EGFR, phosphorylated EGFR ( EGFR-P ), EGR1, FOS, and β-actin ( ACTB ) of whole cell lysates derived from A431 cells propagated in serum-containing media after transduction

    Techniques Used: Expressing, Western Blot, Derivative Assay, Transduction

    AGR2 determines EGFR cell surface expression. Immunofluorescence with anti-EGFR mAb 528 ( A ) or anti-ITGB1 antibodies ( B ) to label non-permeabilized NCI-H460 and A431 cells transduced with a vector control or shAGR2. The nuclei were labeled with DAPI stain
    Figure Legend Snippet: AGR2 determines EGFR cell surface expression. Immunofluorescence with anti-EGFR mAb 528 ( A ) or anti-ITGB1 antibodies ( B ) to label non-permeabilized NCI-H460 and A431 cells transduced with a vector control or shAGR2. The nuclei were labeled with DAPI stain

    Techniques Used: Expressing, Immunofluorescence, Transduction, Plasmid Preparation, Labeling, Staining

    17) Product Images from "Beclin-1-interacting autophagy protein Atg14L targets the SNARE-associated protein Snapin to coordinate endocytic trafficking"

    Article Title: Beclin-1-interacting autophagy protein Atg14L targets the SNARE-associated protein Snapin to coordinate endocytic trafficking

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.100339

    Atg14L facilitates endolysosomal trafficking through its interaction with Snapin. ( A ) Overexpressing Snapin in atg14l knockdown cells fails to promote EGFR degradation. HeLa cells stably expressing atg14l -specific or non-silencing control shRNAmir were
    Figure Legend Snippet: Atg14L facilitates endolysosomal trafficking through its interaction with Snapin. ( A ) Overexpressing Snapin in atg14l knockdown cells fails to promote EGFR degradation. HeLa cells stably expressing atg14l -specific or non-silencing control shRNAmir were

    Techniques Used: Stable Transfection, Expressing

    Snapin promotes endocytic trafficking. ( A ) Depleting Snapin attenuates EGFR degradation. Snapin WT and KO MEFs were treated with EGF (200 ng/ml) for the indicated time periods and WCEs were subjected to IB with anti-EGFR, anti-Snapin or anti-β-Actin
    Figure Legend Snippet: Snapin promotes endocytic trafficking. ( A ) Depleting Snapin attenuates EGFR degradation. Snapin WT and KO MEFs were treated with EGF (200 ng/ml) for the indicated time periods and WCEs were subjected to IB with anti-EGFR, anti-Snapin or anti-β-Actin

    Techniques Used:

    18) Product Images from "EGF receptor kinase suppresses ciliogenesis through activation of USP8 deubiquitinase"

    Article Title: EGF receptor kinase suppresses ciliogenesis through activation of USP8 deubiquitinase

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03117-y

    EGFR contributes cell cycle progression through USP8-trihocplein pathway-mediated cilia suppression. a–c Twenty-four hours after transfection with control or IFT20 siRNA, RPE1 cells were further transfected with control or EGFR siRNA (#1 or #2) and then cultured for 48 h in normal medium (10% FBS). The cells were analyzed by immunoblotting with indicated antibodies ( a ), and immunofluorescence staining with anti-acetylated-tubulin and anti-cyclin A to evaluate percentages of ciliated cell ( b ) and cyclin A-positive cell ( c ), respectively. Scale bars, 20 μm. d TetOn-RPE1 FLAG-USP8 (WT, Y717F/Y810F, Y717E/Y810E, C786S) cells were transfected with control or EGFR siRNA (#1) and then cultured for 48 h in the presence or absence of Dox (10 ng ml −1 ). Immunoblotting analysis with indicated antibodies (left) and percentages of ciliated cell (right) are shown. e Proposed model: normalized intensities of trichoplein/GAPDH in a and d are shown as mean from three independent biological replicates. Graphs represent mean ± SD from three independent experiments ( n > 200 each). ** p
    Figure Legend Snippet: EGFR contributes cell cycle progression through USP8-trihocplein pathway-mediated cilia suppression. a–c Twenty-four hours after transfection with control or IFT20 siRNA, RPE1 cells were further transfected with control or EGFR siRNA (#1 or #2) and then cultured for 48 h in normal medium (10% FBS). The cells were analyzed by immunoblotting with indicated antibodies ( a ), and immunofluorescence staining with anti-acetylated-tubulin and anti-cyclin A to evaluate percentages of ciliated cell ( b ) and cyclin A-positive cell ( c ), respectively. Scale bars, 20 μm. d TetOn-RPE1 FLAG-USP8 (WT, Y717F/Y810F, Y717E/Y810E, C786S) cells were transfected with control or EGFR siRNA (#1) and then cultured for 48 h in the presence or absence of Dox (10 ng ml −1 ). Immunoblotting analysis with indicated antibodies (left) and percentages of ciliated cell (right) are shown. e Proposed model: normalized intensities of trichoplein/GAPDH in a and d are shown as mean from three independent biological replicates. Graphs represent mean ± SD from three independent experiments ( n > 200 each). ** p

    Techniques Used: Transfection, Cell Culture, Immunofluorescence, Staining

    EGFR directly phosphorylates USP8 on Tyr-717 and Tyr-810 to elevate its DUB activity. a Anti-EGFR immunoprecipitates from serum-fed (serum: + ) or -starved (serum: -) RPE1 cell lysates were analyzed by anti-USP8 and anti-EGFR immunoblotting. b Bacterially purified GST-USP8 was incubated with purified GST-EGFR (669–1210 aa) in the presence or absence of 1 μM PD153035 for 15 min at 30 °C. c Bacterially purified non-tagged USP8 variants (WT, Y717F/Y810F, Y717F, and Y810F) were incubated with or without GST-EGFR (669–1210 aa) for 15 min at 30 °C, and then incubated with ubiquitin oligomers (Ub 3–7 ) for 15 min at 37 °C. d TetOn-RPE1 FLAG-USP8 (WT and Y717F/Y810F) cells treated with 10 ng ml −1 of Dox (serum: + ) were subjected to 24 h serum starvation (serum: −). Anti-pY and anti-FLAG immunoblotting of anti-FLAG immunoprecipitates are shown. e , f TetOn-RPE1 FLAG-USP8 (WT and Y717F/Y810F; YF) cells were transfected with control or USP8 siRNA (#1), and then cultured for 48 h in the presence of indicated concentration of Dox. Representative confocal images of acetylated-tubulin (green), FLAG (red) and DAPI (blue) are shown in e . Scale bar, 20 μm. Normalized intensities of trichoplein/GAPDH are calculated by immunoblotting ( f ) and shown as mean from three independent biological replicates. Percentages of ciliated cell (mean ± SD from three independent experiments, n > 200 each) are shown in f . Normalized intensities of trichoplein/GAPDH are shown as mean from three independent biological replicates. ** p
    Figure Legend Snippet: EGFR directly phosphorylates USP8 on Tyr-717 and Tyr-810 to elevate its DUB activity. a Anti-EGFR immunoprecipitates from serum-fed (serum: + ) or -starved (serum: -) RPE1 cell lysates were analyzed by anti-USP8 and anti-EGFR immunoblotting. b Bacterially purified GST-USP8 was incubated with purified GST-EGFR (669–1210 aa) in the presence or absence of 1 μM PD153035 for 15 min at 30 °C. c Bacterially purified non-tagged USP8 variants (WT, Y717F/Y810F, Y717F, and Y810F) were incubated with or without GST-EGFR (669–1210 aa) for 15 min at 30 °C, and then incubated with ubiquitin oligomers (Ub 3–7 ) for 15 min at 37 °C. d TetOn-RPE1 FLAG-USP8 (WT and Y717F/Y810F) cells treated with 10 ng ml −1 of Dox (serum: + ) were subjected to 24 h serum starvation (serum: −). Anti-pY and anti-FLAG immunoblotting of anti-FLAG immunoprecipitates are shown. e , f TetOn-RPE1 FLAG-USP8 (WT and Y717F/Y810F; YF) cells were transfected with control or USP8 siRNA (#1), and then cultured for 48 h in the presence of indicated concentration of Dox. Representative confocal images of acetylated-tubulin (green), FLAG (red) and DAPI (blue) are shown in e . Scale bar, 20 μm. Normalized intensities of trichoplein/GAPDH are calculated by immunoblotting ( f ) and shown as mean from three independent biological replicates. Percentages of ciliated cell (mean ± SD from three independent experiments, n > 200 each) are shown in f . Normalized intensities of trichoplein/GAPDH are shown as mean from three independent biological replicates. ** p

    Techniques Used: Activity Assay, Purification, Incubation, Transfection, Cell Culture, Concentration Assay

    19) Product Images from "EGF receptor kinase suppresses ciliogenesis through activation of USP8 deubiquitinase"

    Article Title: EGF receptor kinase suppresses ciliogenesis through activation of USP8 deubiquitinase

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03117-y

    EGFR contributes cell cycle progression through USP8-trihocplein pathway-mediated cilia suppression. a–c Twenty-four hours after transfection with control or IFT20 siRNA, RPE1 cells were further transfected with control or EGFR siRNA (#1 or #2) and then cultured for 48 h in normal medium (10% FBS). The cells were analyzed by immunoblotting with indicated antibodies ( a ), and immunofluorescence staining with anti-acetylated-tubulin and anti-cyclin A to evaluate percentages of ciliated cell ( b ) and cyclin A-positive cell ( c ), respectively. Scale bars, 20 μm. d TetOn-RPE1 FLAG-USP8 (WT, Y717F/Y810F, Y717E/Y810E, C786S) cells were transfected with control or EGFR siRNA (#1) and then cultured for 48 h in the presence or absence of Dox (10 ng ml −1 ). Immunoblotting analysis with indicated antibodies (left) and percentages of ciliated cell (right) are shown. e Proposed model: normalized intensities of trichoplein/GAPDH in a and d are shown as mean from three independent biological replicates. Graphs represent mean ± SD from three independent experiments ( n > 200 each). ** p
    Figure Legend Snippet: EGFR contributes cell cycle progression through USP8-trihocplein pathway-mediated cilia suppression. a–c Twenty-four hours after transfection with control or IFT20 siRNA, RPE1 cells were further transfected with control or EGFR siRNA (#1 or #2) and then cultured for 48 h in normal medium (10% FBS). The cells were analyzed by immunoblotting with indicated antibodies ( a ), and immunofluorescence staining with anti-acetylated-tubulin and anti-cyclin A to evaluate percentages of ciliated cell ( b ) and cyclin A-positive cell ( c ), respectively. Scale bars, 20 μm. d TetOn-RPE1 FLAG-USP8 (WT, Y717F/Y810F, Y717E/Y810E, C786S) cells were transfected with control or EGFR siRNA (#1) and then cultured for 48 h in the presence or absence of Dox (10 ng ml −1 ). Immunoblotting analysis with indicated antibodies (left) and percentages of ciliated cell (right) are shown. e Proposed model: normalized intensities of trichoplein/GAPDH in a and d are shown as mean from three independent biological replicates. Graphs represent mean ± SD from three independent experiments ( n > 200 each). ** p

    Techniques Used: Transfection, Cell Culture, Immunofluorescence, Staining

    EGFR directly phosphorylates USP8 on Tyr-717 and Tyr-810 to elevate its DUB activity. a Anti-EGFR immunoprecipitates from serum-fed (serum: + ) or -starved (serum: -) RPE1 cell lysates were analyzed by anti-USP8 and anti-EGFR immunoblotting. b Bacterially purified GST-USP8 was incubated with purified GST-EGFR (669–1210 aa) in the presence or absence of 1 μM PD153035 for 15 min at 30 °C. c Bacterially purified non-tagged USP8 variants (WT, Y717F/Y810F, Y717F, and Y810F) were incubated with or without GST-EGFR (669–1210 aa) for 15 min at 30 °C, and then incubated with ubiquitin oligomers (Ub 3–7 ) for 15 min at 37 °C. d TetOn-RPE1 FLAG-USP8 (WT and Y717F/Y810F) cells treated with 10 ng ml −1 of Dox (serum: + ) were subjected to 24 h serum starvation (serum: −). Anti-pY and anti-FLAG immunoblotting of anti-FLAG immunoprecipitates are shown. e , f TetOn-RPE1 FLAG-USP8 (WT and Y717F/Y810F; YF) cells were transfected with control or USP8 siRNA (#1), and then cultured for 48 h in the presence of indicated concentration of Dox. Representative confocal images of acetylated-tubulin (green), FLAG (red) and DAPI (blue) are shown in e . Scale bar, 20 μm. Normalized intensities of trichoplein/GAPDH are calculated by immunoblotting ( f ) and shown as mean from three independent biological replicates. Percentages of ciliated cell (mean ± SD from three independent experiments, n > 200 each) are shown in f . Normalized intensities of trichoplein/GAPDH are shown as mean from three independent biological replicates. ** p
    Figure Legend Snippet: EGFR directly phosphorylates USP8 on Tyr-717 and Tyr-810 to elevate its DUB activity. a Anti-EGFR immunoprecipitates from serum-fed (serum: + ) or -starved (serum: -) RPE1 cell lysates were analyzed by anti-USP8 and anti-EGFR immunoblotting. b Bacterially purified GST-USP8 was incubated with purified GST-EGFR (669–1210 aa) in the presence or absence of 1 μM PD153035 for 15 min at 30 °C. c Bacterially purified non-tagged USP8 variants (WT, Y717F/Y810F, Y717F, and Y810F) were incubated with or without GST-EGFR (669–1210 aa) for 15 min at 30 °C, and then incubated with ubiquitin oligomers (Ub 3–7 ) for 15 min at 37 °C. d TetOn-RPE1 FLAG-USP8 (WT and Y717F/Y810F) cells treated with 10 ng ml −1 of Dox (serum: + ) were subjected to 24 h serum starvation (serum: −). Anti-pY and anti-FLAG immunoblotting of anti-FLAG immunoprecipitates are shown. e , f TetOn-RPE1 FLAG-USP8 (WT and Y717F/Y810F; YF) cells were transfected with control or USP8 siRNA (#1), and then cultured for 48 h in the presence of indicated concentration of Dox. Representative confocal images of acetylated-tubulin (green), FLAG (red) and DAPI (blue) are shown in e . Scale bar, 20 μm. Normalized intensities of trichoplein/GAPDH are calculated by immunoblotting ( f ) and shown as mean from three independent biological replicates. Percentages of ciliated cell (mean ± SD from three independent experiments, n > 200 each) are shown in f . Normalized intensities of trichoplein/GAPDH are shown as mean from three independent biological replicates. ** p

    Techniques Used: Activity Assay, Purification, Incubation, Transfection, Cell Culture, Concentration Assay

    20) Product Images from "Cell adhesion defines the topology of endocytosis and signaling"

    Article Title: Cell adhesion defines the topology of endocytosis and signaling

    Journal: The EMBO Journal

    doi: 10.1002/embj.201385284

    A – C MIPs and merged images of single crossbow-shaped micropatterned cells (upper panel), and corresponding 3D density maps of the 50% probability contours for n cells (lower panel). Density maps are shown in two different views. Fluorescently marked Tfn and TfnR (without permeabilization) after 1 min of ligand addition (A), fluorescently marked EGF and EGFR (without permeabilization) after 1 min of ligand addition (B) and immunostained clathrin heavy chain and AP2 (C) are shown. Scale bars, 10 μm. D Immunoblot of cells transfected with siRNA against luciferase or clathrin heavy chain (CHC) for 72 h. E Normalized count of Tfn and EGF structures per cell after 72 h of transfection with siRNA against luciferase (control) or clathrin heavy chain (CHC). Results are from three independent experiments and 80 cells per condition. F Normalized count of EGF structures per cell after 1 h of treatment with DMSO or filipin. Results are from two independent experiments and 54 cells per condition. G Co-localization analysis of EGF at different concentrations with clathrin, for ≥ 40 cells per condition from two independent experiments. Data information: In (E), (F) and (G), error bars are s.d.
    Figure Legend Snippet: A – C MIPs and merged images of single crossbow-shaped micropatterned cells (upper panel), and corresponding 3D density maps of the 50% probability contours for n cells (lower panel). Density maps are shown in two different views. Fluorescently marked Tfn and TfnR (without permeabilization) after 1 min of ligand addition (A), fluorescently marked EGF and EGFR (without permeabilization) after 1 min of ligand addition (B) and immunostained clathrin heavy chain and AP2 (C) are shown. Scale bars, 10 μm. D Immunoblot of cells transfected with siRNA against luciferase or clathrin heavy chain (CHC) for 72 h. E Normalized count of Tfn and EGF structures per cell after 72 h of transfection with siRNA against luciferase (control) or clathrin heavy chain (CHC). Results are from three independent experiments and 80 cells per condition. F Normalized count of EGF structures per cell after 1 h of treatment with DMSO or filipin. Results are from two independent experiments and 54 cells per condition. G Co-localization analysis of EGF at different concentrations with clathrin, for ≥ 40 cells per condition from two independent experiments. Data information: In (E), (F) and (G), error bars are s.d.

    Techniques Used: Transfection, Luciferase

    21) Product Images from "Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus"

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1002421

    Netrin-1 enhances HCVpp entry. RNAi-mediated knockdown of EGFR. Huh7.5 cells were transfected with anti-EGFR siRNAs #3 and #4 of a previously described study [ 3 ] and infected at a MOI of 0.1. Cells were collected 3 d post-infection and analyzed by RT-qPCR ( A ) and immunoblotting using an anti-EGFR antibody targeting an extracellular epitope of the protein ( B ) (data are represented as mean ± standard deviation, n = 4, Mann-Whitney test, p
    Figure Legend Snippet: Netrin-1 enhances HCVpp entry. RNAi-mediated knockdown of EGFR. Huh7.5 cells were transfected with anti-EGFR siRNAs #3 and #4 of a previously described study [ 3 ] and infected at a MOI of 0.1. Cells were collected 3 d post-infection and analyzed by RT-qPCR ( A ) and immunoblotting using an anti-EGFR antibody targeting an extracellular epitope of the protein ( B ) (data are represented as mean ± standard deviation, n = 4, Mann-Whitney test, p

    Techniques Used: Transfection, Infection, Quantitative RT-PCR, Standard Deviation, MANN-WHITNEY

    22) Product Images from "In Pancreatic Carcinoma, Dual EGFR/HER2 Targeting with Cetuximab/Trastuzumab Is More Effective than Treatment with Trastuzumab/Erlotinib or Lapatinib Alone: Implication of Receptors' Down-regulation and Dimers' Disruption 1"

    Article Title: In Pancreatic Carcinoma, Dual EGFR/HER2 Targeting with Cetuximab/Trastuzumab Is More Effective than Treatment with Trastuzumab/Erlotinib or Lapatinib Alone: Implication of Receptors' Down-regulation and Dimers' Disruption 1

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

    doi:

    Comparison of the effect of lapatinib (dual HER2/EGFR TKI) and of the 2mAbs therapy on tumor growth in nude mice xenografted subcutaneously with BxPC-3 (A), Capan-1 (B), or SKOV-3 (C) cells and randomized in different groups ( n = 10 per group). At days 14 (Capan-1), 20 (BxPC-3), and 11 (SKOV-3 cells) after graft, mice were treated with lapatinib (100, 200, or 300 mg/kg, daily), the 2mAbs therapy (ratio 1:1; 2 mg/kg of each mAb, twice per week) or sterile PBS (daily). Results are presented as the mean tumor volume of each group. Bars, SD of the mean. C indicates cetuximab; Lap, lapatinib; T, trastuzumab.
    Figure Legend Snippet: Comparison of the effect of lapatinib (dual HER2/EGFR TKI) and of the 2mAbs therapy on tumor growth in nude mice xenografted subcutaneously with BxPC-3 (A), Capan-1 (B), or SKOV-3 (C) cells and randomized in different groups ( n = 10 per group). At days 14 (Capan-1), 20 (BxPC-3), and 11 (SKOV-3 cells) after graft, mice were treated with lapatinib (100, 200, or 300 mg/kg, daily), the 2mAbs therapy (ratio 1:1; 2 mg/kg of each mAb, twice per week) or sterile PBS (daily). Results are presented as the mean tumor volume of each group. Bars, SD of the mean. C indicates cetuximab; Lap, lapatinib; T, trastuzumab.

    Techniques Used: Mouse Assay

    Effect of the 2mAbs (cetuximab/trastuzumab) therapy, of erlotinib plus trastuzumab, or of lapatinib alone on receptor heterodimerization and homodimerization using the antibody-based TR-FRET assay. (A) Quantification of EGFR/HER2 heterodimers in Capan-1 and BxPC-3 cells. (B) BxPC-3 cells were treated with increasing concentrations of cetuximab/trastuzumab (ratio 1:1), erlotinib and/or trastuzumab, or lapatinib for 10 minutes and then fixed in 10% formalin. Fixed cells were then incubated with d2-labeled anti-EGFR and lumi4-terbium-labeled anti-HER2 mAbs (directed against different epitopes than the ones recognized by cetuximab and trastuzumab) for 6 hours. Cells were washed, and the TR-FRET signals were measured at 665 nm in the TRF mode (60- µ s delay, 400- µ s integration) on 337 nm excitation to estimate the dimer level. The TR-FRET signal was expressed as ΔF665 (%) and then as the dimer percentage (see Materials and Methods). (C) Effect of the different therapies on EGFR or (D) HER2 homodimer formation. Data are mean ± SEM of three independent experiments performed in triplicate. **** P
    Figure Legend Snippet: Effect of the 2mAbs (cetuximab/trastuzumab) therapy, of erlotinib plus trastuzumab, or of lapatinib alone on receptor heterodimerization and homodimerization using the antibody-based TR-FRET assay. (A) Quantification of EGFR/HER2 heterodimers in Capan-1 and BxPC-3 cells. (B) BxPC-3 cells were treated with increasing concentrations of cetuximab/trastuzumab (ratio 1:1), erlotinib and/or trastuzumab, or lapatinib for 10 minutes and then fixed in 10% formalin. Fixed cells were then incubated with d2-labeled anti-EGFR and lumi4-terbium-labeled anti-HER2 mAbs (directed against different epitopes than the ones recognized by cetuximab and trastuzumab) for 6 hours. Cells were washed, and the TR-FRET signals were measured at 665 nm in the TRF mode (60- µ s delay, 400- µ s integration) on 337 nm excitation to estimate the dimer level. The TR-FRET signal was expressed as ΔF665 (%) and then as the dimer percentage (see Materials and Methods). (C) Effect of the different therapies on EGFR or (D) HER2 homodimer formation. Data are mean ± SEM of three independent experiments performed in triplicate. **** P

    Techniques Used: Incubation, Labeling

    (A) The 2mAbs therapy induces long-term EGFR and HER2 down-regulation and inhibition of AKT phosphorylation in Capan-1 pancreatic carcinoma cells. Mice bearing Capan-1 xenografts were treated with the 2mAbs (cetuximab/trastuzumab) therapy (ratio 1:1; 2 mg/kg of each mAb, twice a week), erlotinib (100 mg/kg, daily) plus trastuzumab (2 mg/kg, twice per week), or lapatinib (300 mg/kg, daily). Tumors were resected at day 2, 7, or 15 after the beginning of treatment. Cell lysates were analyzed by Western blot analysis for EGFR and HER2, total AKT, phosphorylated AKT, total MAPK, and phosphorylated MAPK expression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control. C + T indicates 2mAbs. (B) The 2mAbs therapy induces long-term inhibition of the overall tyrosine phosphorylation profile in Capan-1 tumor cells, whereas the tested TKIs did not. Mice xenografted with Capan-1 pancreatic carcinoma cells were treated with either the two mAbs therapy (ratio 1:1; 2 mg/kg of each mAb, twice per week), erlotinib (100 mg/kg, daily) plus trastuzumab (2 mg/kg, twice per day), or lapatinib (300 mg/kg, daily). Tumors were resected at day 2, 3, or 7 after beginning the mAbs therapy and at 1 hour, 6 hours, 7 days, and 15 days after the beginning of the treatment with erlotinib/trastuzumab or lapatinib. Cell lysates were analyzed by Western blot analysis for tyrosine phosphorylation (P-Tyr). C + T indicates cetuximab/trastuzumab; E + T, erlotinib/trastuzumab.
    Figure Legend Snippet: (A) The 2mAbs therapy induces long-term EGFR and HER2 down-regulation and inhibition of AKT phosphorylation in Capan-1 pancreatic carcinoma cells. Mice bearing Capan-1 xenografts were treated with the 2mAbs (cetuximab/trastuzumab) therapy (ratio 1:1; 2 mg/kg of each mAb, twice a week), erlotinib (100 mg/kg, daily) plus trastuzumab (2 mg/kg, twice per week), or lapatinib (300 mg/kg, daily). Tumors were resected at day 2, 7, or 15 after the beginning of treatment. Cell lysates were analyzed by Western blot analysis for EGFR and HER2, total AKT, phosphorylated AKT, total MAPK, and phosphorylated MAPK expression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control. C + T indicates 2mAbs. (B) The 2mAbs therapy induces long-term inhibition of the overall tyrosine phosphorylation profile in Capan-1 tumor cells, whereas the tested TKIs did not. Mice xenografted with Capan-1 pancreatic carcinoma cells were treated with either the two mAbs therapy (ratio 1:1; 2 mg/kg of each mAb, twice per week), erlotinib (100 mg/kg, daily) plus trastuzumab (2 mg/kg, twice per day), or lapatinib (300 mg/kg, daily). Tumors were resected at day 2, 3, or 7 after beginning the mAbs therapy and at 1 hour, 6 hours, 7 days, and 15 days after the beginning of the treatment with erlotinib/trastuzumab or lapatinib. Cell lysates were analyzed by Western blot analysis for tyrosine phosphorylation (P-Tyr). C + T indicates cetuximab/trastuzumab; E + T, erlotinib/trastuzumab.

    Techniques Used: Inhibition, Mouse Assay, Western Blot, Expressing

    23) Product Images from "TSG101 interaction with HRS mediates endosomal trafficking and receptor down-regulation"

    Article Title: TSG101 interaction with HRS mediates endosomal trafficking and receptor down-regulation

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

    doi: 10.1073/pnas.0932599100

    Assay of ligand-induced EGF receptor degradation. ( A ) Effects of HRS mutations on EGFR degradation. HeLa cells (in a six-well plate) were made deficient in HRS by transfection of 50 nM Hrs-siRNA ( a ) for 24 h. The cells were then transfected with Flag-tagged HRS expression plasmids (siRNA-resistant) (50 ng per well) or control DNA (pBi-Luc) along with pCDNA3-EGFR (200 ng per well). Twenty-four hours after plasmid transfection the cells was starved in Opti-MEM medium for 1 h and then either mock-treated or induced with 150 ng/ml EGF for 90 min. Proteins from cell lysates were separated on SDS-8% polyacrylamide gel, electrotransferred, and immunoblotted with anti-EGFR, antitubulin, and anti-Flag antibodies. EGFR signals were quantitated by x-ray film densitometry (normalized against tubulin signals). Data from three independent experiments were averaged, and percentages of undegraded EGFR upon EGF induction were plotted. ( B ) Effects of TSG101 mutations on EGFR degradation. HeLa cells were made deficient in TSG101 by transfection of 100 nM TSG101 antisense (AS) RNA oligonucleotide ( a ). Cells were then transfected with various TSG101 expression constructs (resistant to antisense inhibition) (200 ng per well) or control DNA along with pCDNA3-EGFR (200 ng per well). Assays were performed as in A .
    Figure Legend Snippet: Assay of ligand-induced EGF receptor degradation. ( A ) Effects of HRS mutations on EGFR degradation. HeLa cells (in a six-well plate) were made deficient in HRS by transfection of 50 nM Hrs-siRNA ( a ) for 24 h. The cells were then transfected with Flag-tagged HRS expression plasmids (siRNA-resistant) (50 ng per well) or control DNA (pBi-Luc) along with pCDNA3-EGFR (200 ng per well). Twenty-four hours after plasmid transfection the cells was starved in Opti-MEM medium for 1 h and then either mock-treated or induced with 150 ng/ml EGF for 90 min. Proteins from cell lysates were separated on SDS-8% polyacrylamide gel, electrotransferred, and immunoblotted with anti-EGFR, antitubulin, and anti-Flag antibodies. EGFR signals were quantitated by x-ray film densitometry (normalized against tubulin signals). Data from three independent experiments were averaged, and percentages of undegraded EGFR upon EGF induction were plotted. ( B ) Effects of TSG101 mutations on EGFR degradation. HeLa cells were made deficient in TSG101 by transfection of 100 nM TSG101 antisense (AS) RNA oligonucleotide ( a ). Cells were then transfected with various TSG101 expression constructs (resistant to antisense inhibition) (200 ng per well) or control DNA along with pCDNA3-EGFR (200 ng per well). Assays were performed as in A .

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Construct, Inhibition

    EGFR colocalization with early and late endosomal markers. ( A and B ) Control naïve HeLa cells. ( C ) HeLa cells with mutant TSG101 were made by first transfection of TSG101-antisensed RNA oligo and subsequent transfection of a mutant TSG101 construct (M95A). ( D ) HeLa cells with mutant HRS were made by transfection of HRS siRNA and subsequent transfection of a mutant HRS construct (HRS-LSAL). Cells were either mock-treated ( A ) or induced ( B–D ) with EGF (150 ng/ml) for 60 min. Cells were then fixed, permeabilized, and immunostained for EGFR and EEA1 (or CD63), and imaged under a deconvolution fluorescence microscope as described in Materials and Methods.
    Figure Legend Snippet: EGFR colocalization with early and late endosomal markers. ( A and B ) Control naïve HeLa cells. ( C ) HeLa cells with mutant TSG101 were made by first transfection of TSG101-antisensed RNA oligo and subsequent transfection of a mutant TSG101 construct (M95A). ( D ) HeLa cells with mutant HRS were made by transfection of HRS siRNA and subsequent transfection of a mutant HRS construct (HRS-LSAL). Cells were either mock-treated ( A ) or induced ( B–D ) with EGF (150 ng/ml) for 60 min. Cells were then fixed, permeabilized, and immunostained for EGFR and EEA1 (or CD63), and imaged under a deconvolution fluorescence microscope as described in Materials and Methods.

    Techniques Used: Mutagenesis, Transfection, Construct, Fluorescence, Microscopy

    Effects of TSG101/HRS interaction on accumulation of ubiquitinated EGFR. HeLa cell (in six-well plates) were first mock-treated (lanes 1 and 2), or transfected with 100 nM TSG101 antisense RNA oligonucleotide (lanes 3 and 4) or 50 nM Hrs-siRNA (lanes 5 and 6) for 24 h. The cells were then transfected with various TSG101 or HRS expression constructs (resistant to antisense inhibition) along with pCDNA3-EGFR (200 ng per well). Twenty-four hours after plasmid transfection the cells were starved and induced with EGF (150 ng/ml) for 90 min. A small portion of cell lysates was analyzed by anti-EGFR Western blot, and the remaining lysates were immunoprecipitated (IP) for EGFR. IP samples were Western-blotted with anti-EGFR and antiubiquitin antibodies.
    Figure Legend Snippet: Effects of TSG101/HRS interaction on accumulation of ubiquitinated EGFR. HeLa cell (in six-well plates) were first mock-treated (lanes 1 and 2), or transfected with 100 nM TSG101 antisense RNA oligonucleotide (lanes 3 and 4) or 50 nM Hrs-siRNA (lanes 5 and 6) for 24 h. The cells were then transfected with various TSG101 or HRS expression constructs (resistant to antisense inhibition) along with pCDNA3-EGFR (200 ng per well). Twenty-four hours after plasmid transfection the cells were starved and induced with EGF (150 ng/ml) for 90 min. A small portion of cell lysates was analyzed by anti-EGFR Western blot, and the remaining lysates were immunoprecipitated (IP) for EGFR. IP samples were Western-blotted with anti-EGFR and antiubiquitin antibodies.

    Techniques Used: Transfection, Expressing, Construct, Inhibition, Plasmid Preparation, Western Blot, Immunoprecipitation

    24) Product Images from "Beclin 1 Is Required for Neuron Viability and Regulates Endosome Pathways via the UVRAG-VPS34 Complex"

    Article Title: Beclin 1 Is Required for Neuron Viability and Regulates Endosome Pathways via the UVRAG-VPS34 Complex

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004626

    Beclin 1 deficient HeLa cells and MEFs display decreased endocytosis. A . Hela cells treated with indicated siRNA (siCon is siControl; siBec is siBeclin 1) were treated with DMSO or Bafilomycin A1 (BafA). Representative composite fluorescence plots and normalized quantification of mean fluorescence intensity of 10,000 cells were determined from three separate experiments. Mean pHrodo dextran (PE-A) fluorescence; bars represent mean +/− s.e.m. (n = 3). DMSO pHrodo dextran siCon vs. siBec one sample t-test (two-tailed) p = 0.0283; BafA pHrodo dextran siCon vs. siBec one-tailed t-test p = 0.0246. B . Mean LysoSensor (Am Cyan-A) fluorescence intensity; bars represent mean +/− s.e.m. (n = 3). LysoSensor siCon vs. siBec one sample t-test (two-tailed) p = 0.0029. C . Anti-UVRAG, -Beclin 1, -Actin and –LC3 blots of HeLa cells lysed after indicated siRNA knock-down. Asterix indicates unspecific band. D . Receptor-mediated endocytosis as measured by EGFR internalization is inhibited in Becn1 deficient MEFs and rescued with re-introduction of beclin 1. Anti-EGFR, - UVRAG, -beclin 1 and –actin blots of control or Becn1 deficient MEFs lysed after indicated treatment with EGF. Asterix indicates unspecific band. Quantification of normalized EGFR/actin from 3 separate experiments. Bars represent mean +/− s.e.m. (n = 3); p = 0.0395, 0.0208 (one-tailed t-test).
    Figure Legend Snippet: Beclin 1 deficient HeLa cells and MEFs display decreased endocytosis. A . Hela cells treated with indicated siRNA (siCon is siControl; siBec is siBeclin 1) were treated with DMSO or Bafilomycin A1 (BafA). Representative composite fluorescence plots and normalized quantification of mean fluorescence intensity of 10,000 cells were determined from three separate experiments. Mean pHrodo dextran (PE-A) fluorescence; bars represent mean +/− s.e.m. (n = 3). DMSO pHrodo dextran siCon vs. siBec one sample t-test (two-tailed) p = 0.0283; BafA pHrodo dextran siCon vs. siBec one-tailed t-test p = 0.0246. B . Mean LysoSensor (Am Cyan-A) fluorescence intensity; bars represent mean +/− s.e.m. (n = 3). LysoSensor siCon vs. siBec one sample t-test (two-tailed) p = 0.0029. C . Anti-UVRAG, -Beclin 1, -Actin and –LC3 blots of HeLa cells lysed after indicated siRNA knock-down. Asterix indicates unspecific band. D . Receptor-mediated endocytosis as measured by EGFR internalization is inhibited in Becn1 deficient MEFs and rescued with re-introduction of beclin 1. Anti-EGFR, - UVRAG, -beclin 1 and –actin blots of control or Becn1 deficient MEFs lysed after indicated treatment with EGF. Asterix indicates unspecific band. Quantification of normalized EGFR/actin from 3 separate experiments. Bars represent mean +/− s.e.m. (n = 3); p = 0.0395, 0.0208 (one-tailed t-test).

    Techniques Used: Fluorescence, Two Tailed Test, One-tailed Test

    25) Product Images from "Autophagic flux blockage in alveolar epithelial cells is essential in silica nanoparticle-induced pulmonary fibrosis"

    Article Title: Autophagic flux blockage in alveolar epithelial cells is essential in silica nanoparticle-induced pulmonary fibrosis

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-1340-8

    SiNPs impair lysosomal degradative capacity. a A549 cells transiently expressing CFP-LC3 and YFP-LAMP1 were either starved or treated with SiNPs at 50 μg/mL for 12 h or 24 h. The cells were then fixed and imaged by confocal microscopy. The graph on the right shows the statistics of the colocalization coefficients of LC3-CFP and LAMP1-YFP. Scale bars: 5 μm. b A549 cells treated with 50 μg/mL of SiNPs for 24 h were pre-incubated with 100 ng/mL of EGF for 15 min, and then the EGF was removed. At the indicated time after EGF treatment, the cells were fixed and immunostained with anti-EGFR antibody. Scale bars: 10 μm. c Quantitative analysis of panel B was performed by normalization to the intensity of cells with EGF treatment for 1 h ( n = 30). d The EGFR protein level from the cells treated as in ( b ) was detected by western blot. Data are presented as mean ± SEM, ** P
    Figure Legend Snippet: SiNPs impair lysosomal degradative capacity. a A549 cells transiently expressing CFP-LC3 and YFP-LAMP1 were either starved or treated with SiNPs at 50 μg/mL for 12 h or 24 h. The cells were then fixed and imaged by confocal microscopy. The graph on the right shows the statistics of the colocalization coefficients of LC3-CFP and LAMP1-YFP. Scale bars: 5 μm. b A549 cells treated with 50 μg/mL of SiNPs for 24 h were pre-incubated with 100 ng/mL of EGF for 15 min, and then the EGF was removed. At the indicated time after EGF treatment, the cells were fixed and immunostained with anti-EGFR antibody. Scale bars: 10 μm. c Quantitative analysis of panel B was performed by normalization to the intensity of cells with EGF treatment for 1 h ( n = 30). d The EGFR protein level from the cells treated as in ( b ) was detected by western blot. Data are presented as mean ± SEM, ** P

    Techniques Used: Expressing, Confocal Microscopy, Incubation, Western Blot

    26) Product Images from "Cooperative Interactions of HER-2 and HPV-16 Oncoproteins in the Malignant Transformation of Human Mammary Epithelial Cells 1"

    Article Title: Cooperative Interactions of HER-2 and HPV-16 Oncoproteins in the Malignant Transformation of Human Mammary Epithelial Cells 1

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

    doi:

    Analysis of erbB receptors in MCF-10HER-2 and derivative cell lines. (A) HER-2, EGFR, and HER3 were immunoprecipitated, separated on 7.5% SDS-PAGE, blotted, and probed for P-Tyr. Nonspecific bands (NSB) from the blots are shown to indicate equivalent
    Figure Legend Snippet: Analysis of erbB receptors in MCF-10HER-2 and derivative cell lines. (A) HER-2, EGFR, and HER3 were immunoprecipitated, separated on 7.5% SDS-PAGE, blotted, and probed for P-Tyr. Nonspecific bands (NSB) from the blots are shown to indicate equivalent

    Techniques Used: Immunoprecipitation, SDS Page

    Effects of blocking EGFR on HER-2 activation. (A) MDA-361 cells (containing HER-2 and not EGFR), MDA-468 cells (containing EGFR and not HER-2), and MCF-10HER2/E6E7 cells were treated with DMSO or 0.1, 0.5, or 1.0 µM Iressa. HER-2 and EGFR were
    Figure Legend Snippet: Effects of blocking EGFR on HER-2 activation. (A) MDA-361 cells (containing HER-2 and not EGFR), MDA-468 cells (containing EGFR and not HER-2), and MCF-10HER2/E6E7 cells were treated with DMSO or 0.1, 0.5, or 1.0 µM Iressa. HER-2 and EGFR were

    Techniques Used: Blocking Assay, Activation Assay, Multiple Displacement Amplification

    27) Product Images from "Met interacts with EGFR and Ron in canine osteosarcoma"

    Article Title: Met interacts with EGFR and Ron in canine osteosarcoma

    Journal: Veterinary and comparative oncology

    doi: 10.1111/j.1476-5829.2011.00309.x

    Ron and EGFR are expressed in OSA cell lines and tumour specimens. Total RNA was extracted from primary OSA tumour samples or 15 × 10 6 cells using TRIzol® reagent followed by cDNA synthesis with reverse transcriptase-PCR to detect message
    Figure Legend Snippet: Ron and EGFR are expressed in OSA cell lines and tumour specimens. Total RNA was extracted from primary OSA tumour samples or 15 × 10 6 cells using TRIzol® reagent followed by cDNA synthesis with reverse transcriptase-PCR to detect message

    Techniques Used: Polymerase Chain Reaction

    Met, EGFR and Ron co-associate in canine OSA. Met, EGFR or Ron was immunoprecipitated from canine OSA cell lines followed by western blot analysis for Met, EGFR or Ron to determine interactions between these RTKs. (A and B) Met and EGFR interaction occurred
    Figure Legend Snippet: Met, EGFR and Ron co-associate in canine OSA. Met, EGFR or Ron was immunoprecipitated from canine OSA cell lines followed by western blot analysis for Met, EGFR or Ron to determine interactions between these RTKs. (A and B) Met and EGFR interaction occurred

    Techniques Used: Immunoprecipitation, Western Blot

    28) Product Images from "Mir-452-3p: A Potential Tumor Promoter That Targets the CPEB3/EGFR Axis in Human Hepatocellular Carcinoma"

    Article Title: Mir-452-3p: A Potential Tumor Promoter That Targets the CPEB3/EGFR Axis in Human Hepatocellular Carcinoma

    Journal: Technology in Cancer Research & Treatment

    doi: 10.1177/1533034617735931

    MiR-452-3p modified the downstream signaling pathway of EGFR. The expressions of CPEB3, p21, EGFR, AKT, pAKT, and β-actin in different groups were detected by Western blot. ** P
    Figure Legend Snippet: MiR-452-3p modified the downstream signaling pathway of EGFR. The expressions of CPEB3, p21, EGFR, AKT, pAKT, and β-actin in different groups were detected by Western blot. ** P

    Techniques Used: Modification, Western Blot

    29) Product Images from "Targeted Vault Nanoparticles Engineered with an Endosomolytic Peptide Deliver Biomolecules to the Cytoplasm"

    Article Title: Targeted Vault Nanoparticles Engineered with an Endosomolytic Peptide Deliver Biomolecules to the Cytoplasm

    Journal: ACS nano

    doi: 10.1021/nn2014613

    Escape of pVI-MVP-Z vaults from the endosome. Comparison of A431 cells incubated with either mCherry-INT/CP-MVP-Z vaults prebound to anti-EGFR antibody mCherry-INT/pVI-MVP-Z vaults prebound to anti-EGFR antibody for the indicated times. The nuclei were
    Figure Legend Snippet: Escape of pVI-MVP-Z vaults from the endosome. Comparison of A431 cells incubated with either mCherry-INT/CP-MVP-Z vaults prebound to anti-EGFR antibody mCherry-INT/pVI-MVP-Z vaults prebound to anti-EGFR antibody for the indicated times. The nuclei were

    Techniques Used: Incubation

    Enhanced Co-transfection of DNA delivery through targeted pVI-MVP-Z vault particles. A431 cells were transfected with CaPi Luc DNA (1.5 μg/well) and pVI-MVP-Z vaults prebound to anti-EGFR antibody with the indicated number of vault particles per
    Figure Legend Snippet: Enhanced Co-transfection of DNA delivery through targeted pVI-MVP-Z vault particles. A431 cells were transfected with CaPi Luc DNA (1.5 μg/well) and pVI-MVP-Z vaults prebound to anti-EGFR antibody with the indicated number of vault particles per

    Techniques Used: Cotransfection, Transfection

    30) Product Images from "Epidermal growth factor-induced proliferation of collecting duct cells from Oak Ridge polycystic kidney mice involves activation of Na+/H+ exchanger"

    Article Title: Epidermal growth factor-induced proliferation of collecting duct cells from Oak Ridge polycystic kidney mice involves activation of Na+/H+ exchanger

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00188.2014

    Representative immunofluorescence images of EGFR and NHE-1 in orpk cilia (+) and cilia (−) cells (magnification ×400). A : EGFR (green) is predominantly localized at the intracellular compartment and basolateral membrane in cilia (+) cells
    Figure Legend Snippet: Representative immunofluorescence images of EGFR and NHE-1 in orpk cilia (+) and cilia (−) cells (magnification ×400). A : EGFR (green) is predominantly localized at the intracellular compartment and basolateral membrane in cilia (+) cells

    Techniques Used: Immunofluorescence

    31) Product Images from "A System-wide Approach to Monitor Responses to Synergistic BRAF and EGFR Inhibition in Colorectal Cancer Cells"

    Article Title: A System-wide Approach to Monitor Responses to Synergistic BRAF and EGFR Inhibition in Colorectal Cancer Cells

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.RA117.000486

    Assessment of WiDr CRC cell growth by combination treatments of BRAF i and ERBB or metabolic inhibitors. A , Comparison of mono- and double therapy on WiDr CRC cells growth. All three graphs show inhibition of either EGFR, EGFR/ERBB2 or EGFR/ERBB2/ERBB3 is ineffective as a monotherapy. Moreover, concomitant inhibition of ERBB2 and ERBB3 does not provide further benefit to the synergistic effect of BRAF(V600E) and EGFR inhibitors. B , WiDr cell confluence is measured comparing double and triple treatments. The addition of etomoxir or DCA as third metabolic inhibitors after 96 h does not show additional benefit to the BRAF i +EGFR i treatment.
    Figure Legend Snippet: Assessment of WiDr CRC cell growth by combination treatments of BRAF i and ERBB or metabolic inhibitors. A , Comparison of mono- and double therapy on WiDr CRC cells growth. All three graphs show inhibition of either EGFR, EGFR/ERBB2 or EGFR/ERBB2/ERBB3 is ineffective as a monotherapy. Moreover, concomitant inhibition of ERBB2 and ERBB3 does not provide further benefit to the synergistic effect of BRAF(V600E) and EGFR inhibitors. B , WiDr cell confluence is measured comparing double and triple treatments. The addition of etomoxir or DCA as third metabolic inhibitors after 96 h does not show additional benefit to the BRAF i +EGFR i treatment.

    Techniques Used: Inhibition

    32) Product Images from "Human Cytomegalovirus Requires Epidermal Growth Factor Receptor Signaling To Enter and Initiate the Early Steps in the Establishment of Latency in CD34+ Human Progenitor Cells"

    Article Title: Human Cytomegalovirus Requires Epidermal Growth Factor Receptor Signaling To Enter and Initiate the Early Steps in the Establishment of Latency in CD34+ Human Progenitor Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.01206-16

    EGFR/PI3K signaling is required for HCMV entry into CD34 + HPCs. (A and B) Viral entry assays were performed in CD34 + ). Cells were pretreated with AG1478 (EGFRK inhibitor), LY294002
    Figure Legend Snippet: EGFR/PI3K signaling is required for HCMV entry into CD34 + HPCs. (A and B) Viral entry assays were performed in CD34 + ). Cells were pretreated with AG1478 (EGFRK inhibitor), LY294002

    Techniques Used:

    HCMV trafficking in CD34 + HPCs is enhanced by EGFR signaling activated during viral entry. (A) CD34 + HPCs were infected with HCMV (TB40-UL32-HCMV/E) and treated with DMSO or AG1478 at 30 mpi. The cells were cytospun onto slides at 1 hpi and 4 hpi and
    Figure Legend Snippet: HCMV trafficking in CD34 + HPCs is enhanced by EGFR signaling activated during viral entry. (A) CD34 + HPCs were infected with HCMV (TB40-UL32-HCMV/E) and treated with DMSO or AG1478 at 30 mpi. The cells were cytospun onto slides at 1 hpi and 4 hpi and

    Techniques Used: Infection

    HMCV infection alters the transcription of cellular hematopoietic factors, in part via EGFR signaling. (A and B) CD34 + HPCs were mock infected or HCMV infected for 4 h to allow viral entry and trafficking to the nucleus. (A, top row, and B) At 4 hpi,
    Figure Legend Snippet: HMCV infection alters the transcription of cellular hematopoietic factors, in part via EGFR signaling. (A and B) CD34 + HPCs were mock infected or HCMV infected for 4 h to allow viral entry and trafficking to the nucleus. (A, top row, and B) At 4 hpi,

    Techniques Used: Infection

    HCMV-induced EGFR signaling favors latent over lytic viral gene expression in CD34 + HPCs. (A to C) CD34 + HPCs were mock infected or HCMV infected for 4 h and then treated with control DMSO or AG1478 (EGFRK inhibitor). RNA was isolated for RT-qPCR at 24
    Figure Legend Snippet: HCMV-induced EGFR signaling favors latent over lytic viral gene expression in CD34 + HPCs. (A to C) CD34 + HPCs were mock infected or HCMV infected for 4 h and then treated with control DMSO or AG1478 (EGFRK inhibitor). RNA was isolated for RT-qPCR at 24

    Techniques Used: Expressing, Infection, Isolation, Quantitative RT-PCR

    EGFR is expressed on the surfaces of CD34 + HPCs and is required for HCMV entry. (A) Uninfected CD34 + HPCs were stained with anti-EGFR antibodies and Alexa Fluor 594-conjugated secondary antibodies to detect EGFR expression on the cell surface (red). DAPI
    Figure Legend Snippet: EGFR is expressed on the surfaces of CD34 + HPCs and is required for HCMV entry. (A) Uninfected CD34 + HPCs were stained with anti-EGFR antibodies and Alexa Fluor 594-conjugated secondary antibodies to detect EGFR expression on the cell surface (red). DAPI

    Techniques Used: Staining, Expressing

    Model for early HCMV infection of CD34 + HPCs. (Left) Various treatments used in this study to investigate HCMV-induced EGFR signaling in CD34 + HPCs. Pretreatment with anti-EGFR blocking antibodies was used to block HCMV-EGFR engagement at the cell surface
    Figure Legend Snippet: Model for early HCMV infection of CD34 + HPCs. (Left) Various treatments used in this study to investigate HCMV-induced EGFR signaling in CD34 + HPCs. Pretreatment with anti-EGFR blocking antibodies was used to block HCMV-EGFR engagement at the cell surface

    Techniques Used: Infection, Blocking Assay

    33) Product Images from "Progressive modulation of the human olfactory bulb transcriptome during Alzheimer´s disease evolution: novel insights into the olfactory signaling across proteinopathies"

    Article Title: Progressive modulation of the human olfactory bulb transcriptome during Alzheimer´s disease evolution: novel insights into the olfactory signaling across proteinopathies

    Journal: Oncotarget

    doi: 10.18632/oncotarget.18193

    Olfactory bulb protein expression of EGFR, and STAT3 across AD staging ( A ) Representative Western blot gels to detect olfactory EGFR across AD grading. ( B ) Protein expression of Total STAT3, and active STAT3 (Y705) in the OB during AD progression. Right panels shows histograms of band densities. Data are presented as mean ± SEM from 3 independent OB samples per group. * P
    Figure Legend Snippet: Olfactory bulb protein expression of EGFR, and STAT3 across AD staging ( A ) Representative Western blot gels to detect olfactory EGFR across AD grading. ( B ) Protein expression of Total STAT3, and active STAT3 (Y705) in the OB during AD progression. Right panels shows histograms of band densities. Data are presented as mean ± SEM from 3 independent OB samples per group. * P

    Techniques Used: Expressing, Western Blot

    Olfactory bulb protein expression of EGFR, CREB1, TGF-beta, c-Jun and STAT3 across proteinopathies OB Protein expression was documented by Western blot. ( A ) EGFR expression, ( B ) TGF-beta expression, ( C ) c-Jun expression, ( D ) STAT3/phospho-STAT3 (Y705) expression, and ( E ) CREB/phospho-CREB (S133) expression, in PSP, FTLD, and mixed dementia subjects. Graphs represent histograms of band densities. Data are presented as mean ± SEM from: Controls ( n = 4 cases), PSP ( n = 9 cases), FTLD ( n = 6 cases), and mixed dementia (mix AD VD) ( n = 9 cases). * P
    Figure Legend Snippet: Olfactory bulb protein expression of EGFR, CREB1, TGF-beta, c-Jun and STAT3 across proteinopathies OB Protein expression was documented by Western blot. ( A ) EGFR expression, ( B ) TGF-beta expression, ( C ) c-Jun expression, ( D ) STAT3/phospho-STAT3 (Y705) expression, and ( E ) CREB/phospho-CREB (S133) expression, in PSP, FTLD, and mixed dementia subjects. Graphs represent histograms of band densities. Data are presented as mean ± SEM from: Controls ( n = 4 cases), PSP ( n = 9 cases), FTLD ( n = 6 cases), and mixed dementia (mix AD VD) ( n = 9 cases). * P

    Techniques Used: Expressing, Western Blot

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    Western Blot:

    Article Title: The Methylation of the TSC2 Promoter Underlies the Abnormal Growth of TSC2 Angiomyolipoma-Derived Smooth Muscle Cells
    Article Snippet: .. In the Western blotting experiments to study S6 and Erk phosphorylation, cells were incubated with rapamycin (1 ng/ml) or anti-EGFR antibody (5 μg/ml) for 24 or 48 hours. .. Cells were incubated for 2 hours with IGF-1 (50 ng/ml) with or without LY294002 (20 μmol/L) (Sigma).

    Incubation:

    Article Title: The Methylation of the TSC2 Promoter Underlies the Abnormal Growth of TSC2 Angiomyolipoma-Derived Smooth Muscle Cells
    Article Snippet: .. In the Western blotting experiments to study S6 and Erk phosphorylation, cells were incubated with rapamycin (1 ng/ml) or anti-EGFR antibody (5 μg/ml) for 24 or 48 hours. .. Cells were incubated for 2 hours with IGF-1 (50 ng/ml) with or without LY294002 (20 μmol/L) (Sigma).

    Article Title: Differential expression of folate receptor 1 in medulloblastoma and the correlation with clinicopathological characters and target therapeutic potential
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    other:

    Article Title: Increased sugar uptake promotes oncogenesis via EPAC/RAP1 and O-GlcNAc pathways
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    Article Title: Vemurafenib Inhibits Active PTK6 in PTEN-null Prostate Tumor Cells
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    Millipore mouse monoclonal antibody against egfr
    No time-dependent phosphorylation of <t>EGFR</t> at <t>Y845</t> but binding of PV-IgG to the keratinocyte cell surface. A: In time-dependent ELISA studies no specific phosphorylation of EGFR at Y845 after PV-IgG 5 treatment (+1. AB) was observed when directly compared with secondary antibody controls (−1. AB). No statistical differences were detected between different time points and experimental conditions (+1. AB vs. −1. AB). B: Binding of PV-IgG 5 to HaCaT cells was determined using quantitative immunostaining of HaCaT cells treated with PV-IgG 5 for the indicated times and stained with Cy3-labeled goat anti-human antibody to detect surface-bound PV-IgG. A clear time-dependent binding of PV-IgG to the keratinocyte surface was detected peaking at 1 hour but declining after 2 hours.
    Mouse Monoclonal Antibody Against Egfr, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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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    Millipore rabbit anti egfr
    Loss of USP32 disrupts cargo trafficking and lysosomal proteolysis. a – e Effect of USP32 depletion on ligand-mediated trafficking and degradation of epidermal growth factor (EGF) receptor <t>(EGFR).</t> a Representative confocal z-projections of fixed HeLa cells transfected as indicated, starved and stimulated with 100 ng/mL EGF-555 (white) for 120 min. Perinuclear (PN) and peripheral (PP) insets show overlays of EGF (green) with immunostained CD63 (magenta). b EGF-positive pixel distribution expressed as fractional distance along a straight line from center of nucleus (0) to the PM (1.0). Red lines: mean, n = 2 independent experiments. c Colocalization of EGF with CD63 in PN (left), PP (middle), and overall (right) in control cells (siCtrl, white bars) vs. those depleted of USP32 (siUSP32_2, gray bars), n = 2 independent experiments. d Representative confocal images of fixed HeLa cells transfected as indicated, starved and stimulated with EGF-555 (white) for 120 min. PN and PP insets show overlays of EGF (magenta) with immunostained cathepsin D (green). e Colocalization of EGF with cathepsin D in control cells (siCtrl, white bars) vs. those depleted of USP32 (siUSP32_2, gray bars), n = 3 independent experiments. All colocalization plots report Mander’s overlap quantified from multicell images (black circles). Cell and nuclear boundaries are depicted in dashed magenta and white lines, respectively. Scale bars = 10 μm. f , g Effect of USP32 depletion on ligand-induced degradation of EGFR. f Lysates from HeLa cells transfected as indicated, serum starved, and stimulated with EGF (25 ng/mL) for 0, 30, 60, or 120 min were analyzed by immunoblot against total EGFR (rabbit anti-EGFR) and phosphorylated (pY) EGFR (mouse <t>anti-phosphotyrosine</t> 4G10), with actin as a loading control. g Total (left graph, relative to t = 0) and activated (right graph, pY relative to t = 30) EGFR remaining at 120 min following stimulation in control cells (siCtrl) vs. those depleted of USP32 using different siRNA oligos (siUSP32_2 and siUSP32_3 + 4), n = 3 independent experiments. Bar graphs report mean of independent measurements (black circles), error bars reflect ±s.d. Total number of cells analyzed per condition appears above each bar/scatter. All significance calculated using Student’s t test: * p
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    No time-dependent phosphorylation of EGFR at Y845 but binding of PV-IgG to the keratinocyte cell surface. A: In time-dependent ELISA studies no specific phosphorylation of EGFR at Y845 after PV-IgG 5 treatment (+1. AB) was observed when directly compared with secondary antibody controls (−1. AB). No statistical differences were detected between different time points and experimental conditions (+1. AB vs. −1. AB). B: Binding of PV-IgG 5 to HaCaT cells was determined using quantitative immunostaining of HaCaT cells treated with PV-IgG 5 for the indicated times and stained with Cy3-labeled goat anti-human antibody to detect surface-bound PV-IgG. A clear time-dependent binding of PV-IgG to the keratinocyte surface was detected peaking at 1 hour but declining after 2 hours.

    Journal: The American Journal of Pathology

    Article Title: Pemphigus Vulgaris IgG Cause Loss of Desmoglein-Mediated Adhesion and Keratinocyte Dissociation Independent of Epidermal Growth Factor Receptor

    doi: 10.2353/ajpath.2009.080392

    Figure Lengend Snippet: No time-dependent phosphorylation of EGFR at Y845 but binding of PV-IgG to the keratinocyte cell surface. A: In time-dependent ELISA studies no specific phosphorylation of EGFR at Y845 after PV-IgG 5 treatment (+1. AB) was observed when directly compared with secondary antibody controls (−1. AB). No statistical differences were detected between different time points and experimental conditions (+1. AB vs. −1. AB). B: Binding of PV-IgG 5 to HaCaT cells was determined using quantitative immunostaining of HaCaT cells treated with PV-IgG 5 for the indicated times and stained with Cy3-labeled goat anti-human antibody to detect surface-bound PV-IgG. A clear time-dependent binding of PV-IgG to the keratinocyte surface was detected peaking at 1 hour but declining after 2 hours.

    Article Snippet: Afterward, HaCaT cells were incubated for 30 minutes with 10% normal goat serum and 1% bovine serum albumin in PBS at room temperature and incubated for 16 hours at 4°C with mouse monoclonal antibody directed against Dsg 3 (Zytomed, Berlin, Germany; dilution 1:100 in PBS), mouse monoclonal antibody against EGFR (Millipore; 1:100), rabbit polyclonal antibody against Y845 phospho-EGFR (Abcam, Cambridge, USA; 1:50), or mouse monoclonal cytokeratin 5 antibody (Santa Cruz, Heidelberg, Germany; 1:100).

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Immunostaining, Staining, Labeling

    PV-IgG did not induce phosphorylation of EGFR at Y845. A: ELISA studies revealed phosphorylation of EGFR at Y845 after EGF but not after control IgG treatment. All three PV-IgG tested also caused an apparent Y845 EGFR phosphorylation. These ELISA signals, however, were also detectable when experiments were performed in the absence of Y845 phospho-EGFR primary antibody indicating cross-reactivity of the secondary goat anti-rabbit (garb) antibody with pemphigus IgG bound to the keratinocyte surface ( n = 4 to 6). B: EGF-induced EGFR phosphorylation at Y845 was reduced by EGFR neutralizing antibody LA-1, inhibition of intrinsic EGFR kinase activity by GW2974 and inhibition of c-Src via PP2. In contrast, these inhibitors did not reduce the PV-IgG 1-induced Y845 EGFR phosphorylation signal ( n = 3). C–J: Moreover, immunostaining using a Y845 phospho-EGFR antibody did not reveal PV-IgG-induced Y845 EGFR phosphorylation. HaCaT cells were immuostained for Y845 phospho-EGFR ( C, E, G, I ) and total EGFR ( D, F, H, J ). Under control conditions, HaCaT cells showed no Y845 phospho-EGFR-specific signal ( C ) but were positive for total EGFR ( D ). EGF treatment (1 hour) resulted in Y845 phospho-EGFR immunostaining at cell borders as well as punctuated staining in the cytoplasm ( arrows in E and F ). Following incubation with PV-IgG, Y845 phospho-EGFR staining was detected at cell borders but was absent within the cytoplasm ( G and H ). Similar staining patterns were obtained after PV-IgG treatment when secondary garb or goat anti-human (gah) antibodies were applied in the absence of primary antibodies ( I and J ). Scale bar = 20 μm for all panels. ( n = 3). K: Western blotting revealed that EGF but not PV-IgG caused Y845 EGFR phosphorylation. In controls and following incubation with PV-IgG, no band was detectable at 170 kDa indicating that EGFR was not phosphorylated at Y845 under these conditions. In contrast, EGF treatment resulted in a strong immunosignal at the expected EGFR size (170 kDa). After treatment with PV-IgG, a strong band was visible migrating at 55 kDa. This band likely represented PV-IgG heavy chains in cell lysates as demonstrated by Western blotting using secondary garb or gah antibodies in the absence of primary antibodies ( L ). ( n = 3). M: Western blotting with NHEK lysates confirmed that treatment with EGF but not with PV-IgG resulted in phosphorylation of EGFR at Y845. Total EGFR bands revealed equal sample loading.

    Journal: The American Journal of Pathology

    Article Title: Pemphigus Vulgaris IgG Cause Loss of Desmoglein-Mediated Adhesion and Keratinocyte Dissociation Independent of Epidermal Growth Factor Receptor

    doi: 10.2353/ajpath.2009.080392

    Figure Lengend Snippet: PV-IgG did not induce phosphorylation of EGFR at Y845. A: ELISA studies revealed phosphorylation of EGFR at Y845 after EGF but not after control IgG treatment. All three PV-IgG tested also caused an apparent Y845 EGFR phosphorylation. These ELISA signals, however, were also detectable when experiments were performed in the absence of Y845 phospho-EGFR primary antibody indicating cross-reactivity of the secondary goat anti-rabbit (garb) antibody with pemphigus IgG bound to the keratinocyte surface ( n = 4 to 6). B: EGF-induced EGFR phosphorylation at Y845 was reduced by EGFR neutralizing antibody LA-1, inhibition of intrinsic EGFR kinase activity by GW2974 and inhibition of c-Src via PP2. In contrast, these inhibitors did not reduce the PV-IgG 1-induced Y845 EGFR phosphorylation signal ( n = 3). C–J: Moreover, immunostaining using a Y845 phospho-EGFR antibody did not reveal PV-IgG-induced Y845 EGFR phosphorylation. HaCaT cells were immuostained for Y845 phospho-EGFR ( C, E, G, I ) and total EGFR ( D, F, H, J ). Under control conditions, HaCaT cells showed no Y845 phospho-EGFR-specific signal ( C ) but were positive for total EGFR ( D ). EGF treatment (1 hour) resulted in Y845 phospho-EGFR immunostaining at cell borders as well as punctuated staining in the cytoplasm ( arrows in E and F ). Following incubation with PV-IgG, Y845 phospho-EGFR staining was detected at cell borders but was absent within the cytoplasm ( G and H ). Similar staining patterns were obtained after PV-IgG treatment when secondary garb or goat anti-human (gah) antibodies were applied in the absence of primary antibodies ( I and J ). Scale bar = 20 μm for all panels. ( n = 3). K: Western blotting revealed that EGF but not PV-IgG caused Y845 EGFR phosphorylation. In controls and following incubation with PV-IgG, no band was detectable at 170 kDa indicating that EGFR was not phosphorylated at Y845 under these conditions. In contrast, EGF treatment resulted in a strong immunosignal at the expected EGFR size (170 kDa). After treatment with PV-IgG, a strong band was visible migrating at 55 kDa. This band likely represented PV-IgG heavy chains in cell lysates as demonstrated by Western blotting using secondary garb or gah antibodies in the absence of primary antibodies ( L ). ( n = 3). M: Western blotting with NHEK lysates confirmed that treatment with EGF but not with PV-IgG resulted in phosphorylation of EGFR at Y845. Total EGFR bands revealed equal sample loading.

    Article Snippet: Afterward, HaCaT cells were incubated for 30 minutes with 10% normal goat serum and 1% bovine serum albumin in PBS at room temperature and incubated for 16 hours at 4°C with mouse monoclonal antibody directed against Dsg 3 (Zytomed, Berlin, Germany; dilution 1:100 in PBS), mouse monoclonal antibody against EGFR (Millipore; 1:100), rabbit polyclonal antibody against Y845 phospho-EGFR (Abcam, Cambridge, USA; 1:50), or mouse monoclonal cytokeratin 5 antibody (Santa Cruz, Heidelberg, Germany; 1:100).

    Techniques: Enzyme-linked Immunosorbent Assay, Inhibition, Activity Assay, Immunostaining, Staining, Incubation, Western Blot

    Inhibition of EGFR or c-Src did not prevent PV-IgG-induced loss of Dsg-mediated bead binding in laser tweezer experiments. Dsg 3- (black bars) and Dsg 1 coated beads (white bars) were allowed to settle on the surface of HaCaT cells for 30 minutes before the number of bound beads was counted (control). After application of PV-IgG or EGF, the number of adhering beads was counted again. The number of bound Dsg 3- or Dsg 1-coated beads was not altered after control IgG treatment but reduced by incubation with EGTA (5 mmol/L, 30 minutes) to 41% ± 6% and 22% ± 3%, respectively. EGF did not block Dsg 3- or Dsg 1-mediated binding after incubation for 30 minutes or 24 hours. In contrast, incubation of monolayers and attached beads with PV-IgG 2 for 30 minutes reduced the numbers of Dsg 3 and Dsg 1 beads to 75% ± 6% and 54% ± 4%, respectively. This reduction was unaffected by blocking of EGFR via preincubation with GW2974, or of c-Src via preincubation with PP2. ( n = 6 for each condition).

    Journal: The American Journal of Pathology

    Article Title: Pemphigus Vulgaris IgG Cause Loss of Desmoglein-Mediated Adhesion and Keratinocyte Dissociation Independent of Epidermal Growth Factor Receptor

    doi: 10.2353/ajpath.2009.080392

    Figure Lengend Snippet: Inhibition of EGFR or c-Src did not prevent PV-IgG-induced loss of Dsg-mediated bead binding in laser tweezer experiments. Dsg 3- (black bars) and Dsg 1 coated beads (white bars) were allowed to settle on the surface of HaCaT cells for 30 minutes before the number of bound beads was counted (control). After application of PV-IgG or EGF, the number of adhering beads was counted again. The number of bound Dsg 3- or Dsg 1-coated beads was not altered after control IgG treatment but reduced by incubation with EGTA (5 mmol/L, 30 minutes) to 41% ± 6% and 22% ± 3%, respectively. EGF did not block Dsg 3- or Dsg 1-mediated binding after incubation for 30 minutes or 24 hours. In contrast, incubation of monolayers and attached beads with PV-IgG 2 for 30 minutes reduced the numbers of Dsg 3 and Dsg 1 beads to 75% ± 6% and 54% ± 4%, respectively. This reduction was unaffected by blocking of EGFR via preincubation with GW2974, or of c-Src via preincubation with PP2. ( n = 6 for each condition).

    Article Snippet: Afterward, HaCaT cells were incubated for 30 minutes with 10% normal goat serum and 1% bovine serum albumin in PBS at room temperature and incubated for 16 hours at 4°C with mouse monoclonal antibody directed against Dsg 3 (Zytomed, Berlin, Germany; dilution 1:100 in PBS), mouse monoclonal antibody against EGFR (Millipore; 1:100), rabbit polyclonal antibody against Y845 phospho-EGFR (Abcam, Cambridge, USA; 1:50), or mouse monoclonal cytokeratin 5 antibody (Santa Cruz, Heidelberg, Germany; 1:100).

    Techniques: Inhibition, Binding Assay, Incubation, Blocking Assay

    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

    Loss of USP32 disrupts cargo trafficking and lysosomal proteolysis. a – e Effect of USP32 depletion on ligand-mediated trafficking and degradation of epidermal growth factor (EGF) receptor (EGFR). a Representative confocal z-projections of fixed HeLa cells transfected as indicated, starved and stimulated with 100 ng/mL EGF-555 (white) for 120 min. Perinuclear (PN) and peripheral (PP) insets show overlays of EGF (green) with immunostained CD63 (magenta). b EGF-positive pixel distribution expressed as fractional distance along a straight line from center of nucleus (0) to the PM (1.0). Red lines: mean, n = 2 independent experiments. c Colocalization of EGF with CD63 in PN (left), PP (middle), and overall (right) in control cells (siCtrl, white bars) vs. those depleted of USP32 (siUSP32_2, gray bars), n = 2 independent experiments. d Representative confocal images of fixed HeLa cells transfected as indicated, starved and stimulated with EGF-555 (white) for 120 min. PN and PP insets show overlays of EGF (magenta) with immunostained cathepsin D (green). e Colocalization of EGF with cathepsin D in control cells (siCtrl, white bars) vs. those depleted of USP32 (siUSP32_2, gray bars), n = 3 independent experiments. All colocalization plots report Mander’s overlap quantified from multicell images (black circles). Cell and nuclear boundaries are depicted in dashed magenta and white lines, respectively. Scale bars = 10 μm. f , g Effect of USP32 depletion on ligand-induced degradation of EGFR. f Lysates from HeLa cells transfected as indicated, serum starved, and stimulated with EGF (25 ng/mL) for 0, 30, 60, or 120 min were analyzed by immunoblot against total EGFR (rabbit anti-EGFR) and phosphorylated (pY) EGFR (mouse anti-phosphotyrosine 4G10), with actin as a loading control. g Total (left graph, relative to t = 0) and activated (right graph, pY relative to t = 30) EGFR remaining at 120 min following stimulation in control cells (siCtrl) vs. those depleted of USP32 using different siRNA oligos (siUSP32_2 and siUSP32_3 + 4), n = 3 independent experiments. Bar graphs report mean of independent measurements (black circles), error bars reflect ±s.d. Total number of cells analyzed per condition appears above each bar/scatter. All significance calculated using Student’s t test: * p

    Journal: Nature Communications

    Article Title: USP32 regulates late endosomal transport and recycling through deubiquitylation of Rab7

    doi: 10.1038/s41467-019-09437-x

    Figure Lengend Snippet: Loss of USP32 disrupts cargo trafficking and lysosomal proteolysis. a – e Effect of USP32 depletion on ligand-mediated trafficking and degradation of epidermal growth factor (EGF) receptor (EGFR). a Representative confocal z-projections of fixed HeLa cells transfected as indicated, starved and stimulated with 100 ng/mL EGF-555 (white) for 120 min. Perinuclear (PN) and peripheral (PP) insets show overlays of EGF (green) with immunostained CD63 (magenta). b EGF-positive pixel distribution expressed as fractional distance along a straight line from center of nucleus (0) to the PM (1.0). Red lines: mean, n = 2 independent experiments. c Colocalization of EGF with CD63 in PN (left), PP (middle), and overall (right) in control cells (siCtrl, white bars) vs. those depleted of USP32 (siUSP32_2, gray bars), n = 2 independent experiments. d Representative confocal images of fixed HeLa cells transfected as indicated, starved and stimulated with EGF-555 (white) for 120 min. PN and PP insets show overlays of EGF (magenta) with immunostained cathepsin D (green). e Colocalization of EGF with cathepsin D in control cells (siCtrl, white bars) vs. those depleted of USP32 (siUSP32_2, gray bars), n = 3 independent experiments. All colocalization plots report Mander’s overlap quantified from multicell images (black circles). Cell and nuclear boundaries are depicted in dashed magenta and white lines, respectively. Scale bars = 10 μm. f , g Effect of USP32 depletion on ligand-induced degradation of EGFR. f Lysates from HeLa cells transfected as indicated, serum starved, and stimulated with EGF (25 ng/mL) for 0, 30, 60, or 120 min were analyzed by immunoblot against total EGFR (rabbit anti-EGFR) and phosphorylated (pY) EGFR (mouse anti-phosphotyrosine 4G10), with actin as a loading control. g Total (left graph, relative to t = 0) and activated (right graph, pY relative to t = 30) EGFR remaining at 120 min following stimulation in control cells (siCtrl) vs. those depleted of USP32 using different siRNA oligos (siUSP32_2 and siUSP32_3 + 4), n = 3 independent experiments. Bar graphs report mean of independent measurements (black circles), error bars reflect ±s.d. Total number of cells analyzed per condition appears above each bar/scatter. All significance calculated using Student’s t test: * p

    Article Snippet: The following antibodies were used for detection of endogenous and overexpressed proteins by Western blot analysis in a 1:1000 dilution: mouse anti-USP32 (A-10) in (Santa Cruz Biotechnology, Cat# sc-374465), rabbit anti-VPS26 (Abcam, Cat# ab181352), goat anti-VSP35 (Abcam, Cat# ab10099), mouse anti-VPS35 (Santa Cruz Biotechnology, Cat# sc-374372), mouse anti-CI-M6PR (Abcam, Cat# ab2733), rabbit anti-cathepsin D (Abcam, Cat# ab75852), rabbit anti-EGFR (Millipore, Cat # 06-847), mouse anti-phosphotyrosine (4G10 Millipore, Cat# 05-321), rabbit anti-mGFP , mouse anti-HA (HA.11 (16B12), Covance, Cat# MMS-101R), and anti-mRFP .

    Techniques: Transfection