anti egfr  (Millipore)

 
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    Name:
    Anti Epidermal Growth Factor Receptor antibody
    Description:
    EGFR is a glycoprotein receptor that consists of 3 domains an extracellular domain which modulates ligand binding and receptor dimerization a single transmembrane domain and a cytoplasmic domain EGFR ErbB1 binds to EGF ligands and is known to form homodimers or heterodimers Furthermore EGFR regulates differentiation growth movement and apoptosis in a wide range of cells Sheep anti EGFR recognizes rat mouse dog chicken and human EGF receptors Furthermore the antibody recognizes the internal domain of the receptor molecule and will block the phosphorylation but will not block EGF binding The antibody can be used with formalin fixed paraffin embedded tissue sections
    Catalog Number:
    A204
    Price:
    None
    Applications:
    Sheep anti-EGFR antibody was used to detect EGFR by immunofluorescence in mouse oocyte cumulus cells that were previously fixed in 4% paraformaldehyde
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    Structured Review

    Millipore anti egfr
    Vemurafenib inhibits activation and signaling downstream of <t>PTK6.</t> (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 <t>EGFR</t> 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.
    EGFR is a glycoprotein receptor that consists of 3 domains an extracellular domain which modulates ligand binding and receptor dimerization a single transmembrane domain and a cytoplasmic domain EGFR ErbB1 binds to EGF ligands and is known to form homodimers or heterodimers Furthermore EGFR regulates differentiation growth movement and apoptosis in a wide range of cells Sheep anti EGFR recognizes rat mouse dog chicken and human EGF receptors Furthermore the antibody recognizes the internal domain of the receptor molecule and will block the phosphorylation but will not block EGF binding The antibody can be used with formalin fixed paraffin embedded tissue sections
    https://www.bioz.com/result/anti egfr/product/Millipore
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti egfr - by Bioz Stars, 2021-05
    93/100 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 "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

    3) 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:

    4) 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

    5) 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

    6) 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

    7) 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

    8) 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

    9) 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

    10) 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

    11) 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

    12) 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

    13) 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

    14) 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

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    Article Snippet: Subsequently, blocking of nonspecific binding sites was achieved using 10% normal serum diluted in PBS with 0.3% Triton-X (PBST; Sigma) to aid permeabilization. .. Primary antibodies, goat anti-β-galactosidase (β-gal) (1:200, Biogen, Cambridge, MA), sheep anti-EGFR (1:20; Millipore, Bedford, MA), rabbit anti-Gsh2 (1:2000; ; kind gift of K. Campbell), mouse anti-Pax6 (1:2000; Developmental Studies Hybridoma Bank [DSHB], University of Iowa, Iowa City, IA), rabbit anti-Pax6 (1:1000; Covance Research Products, Berkeley, CA), mouse anti-RC2 (1:20; DSHB), rabbit anti-Tbr1 (1:1000; ; kind gift of R. Hevner) were diluted in 1% normal serum in PBST and incubated overnight at 4 °C. .. Following 3 × 10 min rinses in PBS, the sections were incubated with the appropriate secondary Cy3- (1:200) or fluorescein isothiocyanate- (1:50) conjugated antibodies (serum and secondary antibodies were from Jackson Immunoresearch, West Grove, PA), diluted as for the primary antibodies, in the dark for 2 h at RT.

    Incubation:

    Article Title: Differential Regulation of Telencephalic Pallial–Subpallial Boundary Patterning by Pax6 and Gsh2
    Article Snippet: Subsequently, blocking of nonspecific binding sites was achieved using 10% normal serum diluted in PBS with 0.3% Triton-X (PBST; Sigma) to aid permeabilization. .. Primary antibodies, goat anti-β-galactosidase (β-gal) (1:200, Biogen, Cambridge, MA), sheep anti-EGFR (1:20; Millipore, Bedford, MA), rabbit anti-Gsh2 (1:2000; ; kind gift of K. Campbell), mouse anti-Pax6 (1:2000; Developmental Studies Hybridoma Bank [DSHB], University of Iowa, Iowa City, IA), rabbit anti-Pax6 (1:1000; Covance Research Products, Berkeley, CA), mouse anti-RC2 (1:20; DSHB), rabbit anti-Tbr1 (1:1000; ; kind gift of R. Hevner) were diluted in 1% normal serum in PBST and incubated overnight at 4 °C. .. Following 3 × 10 min rinses in PBS, the sections were incubated with the appropriate secondary Cy3- (1:200) or fluorescein isothiocyanate- (1:50) conjugated antibodies (serum and secondary antibodies were from Jackson Immunoresearch, West Grove, PA), diluted as for the primary antibodies, in the dark for 2 h at RT.

    Article Title: Angiotensin II stimulates fibronectin protein synthesis via a Gβγ/arachidonic acid-dependent pathway
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    SDS Page:

    Article Title: Angiotensin II stimulates fibronectin protein synthesis via a Gβγ/arachidonic acid-dependent pathway
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  • 92
    Millipore sheep anti egfr
    Western blot analysis of <t>EGFR</t> signal. Total EGFR and <t>pEGFR</t> in liver protein lysates from A) APC Min /+ and B) AOM mice, separately for each strain and sex. STD = standard diet, WD = western diet, “+” = AG1478 treatment and “−“ = no AG1478 treatment. Below bar graphs are estimated effects and significance based on linear mixed models.
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    PD168393 inhibited epidermal growth factor receptor <t>(EGFR)</t> phosphorylation of astrocytes after spinal cord injury (SCI) in rats. Spinal cord sections were immunostained for phosphorylated EGFR (pEGFR) and glial fibrillary acid protein <t>(GFAP)</t> at two weeks after SCI (n = 5/group). (A1-C1) GFAP, (A2-C2) pEFGR, and (A3-C3) co-localization of pEGFR and GFAP. Insets in panels (B1-C3) are shown at high magnification in panels (D1-E3) , respectively. Scale bars = 500 μm in (C3) (applies to A1-C3 ); scale bars = 50 μm in (E3) (applies to D1-E3 ). Representative Western blots of pEGFR and EGFR expression (n = 3/group), and β-actin was a loading control (F) . Semi-quantitative measurements were obtained by normalizing to β-actin (G) . * P
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    Millipore anti mpm2 antibody
    Ser77 is phosphorylated by EGF stimulation. (A) Vero cells were transfected with the indicated mutant palladin. Twenty-four hours later, serum-starved cells were stimulated with EGF for 5 min. Mobility shifts were examined by western blotting. (B) Mobility shifts of Δ44-S197G and Δ102-S197G were examined by western blotting. (C) Amino acid sequence from 45 to 102 is indicated. Mobility shift of the indicated mutant palladin was examined by western blotting. (D) Mobility shift of the indicated mutant palladin was examined by western blotting. (E) Vero cells were transfected with either FL-wt or FL-S77.197G palladin. Twenty-four hours later, cells were stimulated with EGF for 5 min. Cells were lysed and immunoprecipited with anti-Myc antibody and blotted with <t>anti-MPM2</t> antibody.
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    Western blot analysis of EGFR signal. Total EGFR and pEGFR in liver protein lysates from A) APC Min /+ and B) AOM mice, separately for each strain and sex. STD = standard diet, WD = western diet, “+” = AG1478 treatment and “−“ = no AG1478 treatment. Below bar graphs are estimated effects and significance based on linear mixed models.

    Journal: PLoS ONE

    Article Title: Efficacy of EGFR Inhibition Is Modulated by Model, Sex, Genetic Background and Diet: Implications for Preclinical Cancer Prevention and Therapy Trials

    doi: 10.1371/journal.pone.0039552

    Figure Lengend Snippet: Western blot analysis of EGFR signal. Total EGFR and pEGFR in liver protein lysates from A) APC Min /+ and B) AOM mice, separately for each strain and sex. STD = standard diet, WD = western diet, “+” = AG1478 treatment and “−“ = no AG1478 treatment. Below bar graphs are estimated effects and significance based on linear mixed models.

    Article Snippet: After transfer to PVDF membranes for one hour, membranes were blocked for one hour in 5% BSA/TBS (0.1% Tween) for rabbit anti-pEGFR 1068 antibody (Cell Signaling) or 5% milk/TBST for sheep anti-EGFR (Millipore) and mouse anti-ACTB (Sigma) antibodies.

    Techniques: Western Blot, Mouse Assay

    Effect Usp18 depletion has on EGFR levels requires endogenous EGFR mRNA. (A and B) EGFR mRNA levels are not affected by depletion of Usp18. SCC2 and COS1 cells, respectively, were transfected with siRNA (150 nM), and the levels of EGFR mRNA were determined using real-time PCR. (C) Usp18 depletion dramatically inhibits translation from native EGFR mRNA but not from EGFR mRNA lacking native UTR sequences. HeLa cells stably expressing EGFR-GFP were treated with Usp18 siRNA and the protein levels of endogenous EGFR and EGFR-GFP were determined using Western blot analysis. (D) EGFR and EGFR-GFP protein levels were quantitated from C and two additional similar experiments with HeLa/EGFR-GFP cells.

    Journal: Molecular Biology of the Cell

    Article Title: RNA Interference Screen Identifies Usp18 as a Regulator of Epidermal Growth Factor Receptor Synthesis

    doi: 10.1091/mbc.E08-08-0880

    Figure Lengend Snippet: Effect Usp18 depletion has on EGFR levels requires endogenous EGFR mRNA. (A and B) EGFR mRNA levels are not affected by depletion of Usp18. SCC2 and COS1 cells, respectively, were transfected with siRNA (150 nM), and the levels of EGFR mRNA were determined using real-time PCR. (C) Usp18 depletion dramatically inhibits translation from native EGFR mRNA but not from EGFR mRNA lacking native UTR sequences. HeLa cells stably expressing EGFR-GFP were treated with Usp18 siRNA and the protein levels of endogenous EGFR and EGFR-GFP were determined using Western blot analysis. (D) EGFR and EGFR-GFP protein levels were quantitated from C and two additional similar experiments with HeLa/EGFR-GFP cells.

    Article Snippet: Monoclonal antibodies to EGFR (#528) were purified from hybridoma cells from American Type Culture Collection (Manassas, VA); antibodies to green fluorescent protein (GFP) and transferrin receptor were from Zymed Laboratories (San Francisco, CA); EGFR (#05-104) was from Millipore (Billerica, MA); α-actinin was from Millipore Bioscience Research Reagents (Temecula, CA) (MAB 1682); ErbB2 for SKBR3 and BT474 cells was from NeoMarkers (Fremont, CA); and ErbB2 for SCC2 cells was from Cell Signaling Technology (Danvers, MA) (29D8).

    Techniques: Transfection, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing, Western Blot

    Multiple Usp18 siRNA oligonucleotides each dramatically reduce Usp18 expression and subsequently promote total cellular down-regulation of EGFR. (A) Usp18 mRNA levels are reduced up to 90% in SCC2 cells after Usp18 siRNA treatment. Nontargeting and Usp18 targeting siRNA oligonucleotides were forward transfected into SCC2 cells twice at 24-h intervals at a concentration of 150 nM. After 72 h, total RNA was isolated from these cells and Usp18 mRNA levels were determined by real-time PCR. The values for each sample were normalized to rRNA and then to nontargeting siRNA values. (B) Depletion of Usp18 from SCC2 cells leads to a 55% reduction in EGFR activation and total cellular EGFR levels. SCC2 cells were transfected with the indicated siRNA oligonucleotides (100 nM). After 72 h, cells were treated with 40 ng/ml EGF for the indicated times. Whole-cell lysates were subjected to Western blot analysis using EGFR 1005 (total EGFR), EGFR pY1068 (phosphorylated EGFR), and α-actinin (loading control) antibodies. All Western blots in this study are representative of three or more experiments. The ratio of activated EGFR to total cellular EGFR was calculated and displayed below the blots. (C) EGFR immunofluorescence reveals that despite lower levels, EGFR cellular distribution does not significantly change after depletion of Usp18. SCC2 cells treated with siRNA were fixed, permeabilized with saponin, and subjected to immunofluorescence with EGFR 528 antibody. All fluorescence intensity settings are identical for the two images. Images were acquired in 20-stack z-series, and the total cellular EGFR signal was quantitated for each cell. Cells treated with Usp18 siRNA had an average 45% decrease in EGFR signal compared with control cells (bar graph). Data were obtained from a minimum of 12 cells. Bars, 10 μm. (D) SCC2 cells depleted of Usp18 have a reduced number of EGF binding sites on the cell surface. After transfection with siRNA, SCC2 cells were placed on ice and incubated with 125 I-EGF for 1 h. The amount of surface-bound 125 I-EGF is expressed as a percentage relative to control cells set to 100%. (E) Depletion of Usp18 from SCC2 cells leads to a 55% reduction in total cellular EGFR levels. SCC2 cells were transfected with the indicated siRNA as in B. Whole-cell lysates were subjected to Western blot analysis by using antibody EGFR 05-104 (recognizing the extracellular domain of EGFR) and an antibody to the transferrin receptor (TransfR).

    Journal: Molecular Biology of the Cell

    Article Title: RNA Interference Screen Identifies Usp18 as a Regulator of Epidermal Growth Factor Receptor Synthesis

    doi: 10.1091/mbc.E08-08-0880

    Figure Lengend Snippet: Multiple Usp18 siRNA oligonucleotides each dramatically reduce Usp18 expression and subsequently promote total cellular down-regulation of EGFR. (A) Usp18 mRNA levels are reduced up to 90% in SCC2 cells after Usp18 siRNA treatment. Nontargeting and Usp18 targeting siRNA oligonucleotides were forward transfected into SCC2 cells twice at 24-h intervals at a concentration of 150 nM. After 72 h, total RNA was isolated from these cells and Usp18 mRNA levels were determined by real-time PCR. The values for each sample were normalized to rRNA and then to nontargeting siRNA values. (B) Depletion of Usp18 from SCC2 cells leads to a 55% reduction in EGFR activation and total cellular EGFR levels. SCC2 cells were transfected with the indicated siRNA oligonucleotides (100 nM). After 72 h, cells were treated with 40 ng/ml EGF for the indicated times. Whole-cell lysates were subjected to Western blot analysis using EGFR 1005 (total EGFR), EGFR pY1068 (phosphorylated EGFR), and α-actinin (loading control) antibodies. All Western blots in this study are representative of three or more experiments. The ratio of activated EGFR to total cellular EGFR was calculated and displayed below the blots. (C) EGFR immunofluorescence reveals that despite lower levels, EGFR cellular distribution does not significantly change after depletion of Usp18. SCC2 cells treated with siRNA were fixed, permeabilized with saponin, and subjected to immunofluorescence with EGFR 528 antibody. All fluorescence intensity settings are identical for the two images. Images were acquired in 20-stack z-series, and the total cellular EGFR signal was quantitated for each cell. Cells treated with Usp18 siRNA had an average 45% decrease in EGFR signal compared with control cells (bar graph). Data were obtained from a minimum of 12 cells. Bars, 10 μm. (D) SCC2 cells depleted of Usp18 have a reduced number of EGF binding sites on the cell surface. After transfection with siRNA, SCC2 cells were placed on ice and incubated with 125 I-EGF for 1 h. The amount of surface-bound 125 I-EGF is expressed as a percentage relative to control cells set to 100%. (E) Depletion of Usp18 from SCC2 cells leads to a 55% reduction in total cellular EGFR levels. SCC2 cells were transfected with the indicated siRNA as in B. Whole-cell lysates were subjected to Western blot analysis by using antibody EGFR 05-104 (recognizing the extracellular domain of EGFR) and an antibody to the transferrin receptor (TransfR).

    Article Snippet: Monoclonal antibodies to EGFR (#528) were purified from hybridoma cells from American Type Culture Collection (Manassas, VA); antibodies to green fluorescent protein (GFP) and transferrin receptor were from Zymed Laboratories (San Francisco, CA); EGFR (#05-104) was from Millipore (Billerica, MA); α-actinin was from Millipore Bioscience Research Reagents (Temecula, CA) (MAB 1682); ErbB2 for SKBR3 and BT474 cells was from NeoMarkers (Fremont, CA); and ErbB2 for SCC2 cells was from Cell Signaling Technology (Danvers, MA) (29D8).

    Techniques: Expressing, Transfection, Concentration Assay, Isolation, Real-time Polymerase Chain Reaction, Activation Assay, Western Blot, Immunofluorescence, Fluorescence, Binding Assay, Incubation

    PD168393 inhibited epidermal growth factor receptor (EGFR) phosphorylation of astrocytes after spinal cord injury (SCI) in rats. Spinal cord sections were immunostained for phosphorylated EGFR (pEGFR) and glial fibrillary acid protein (GFAP) at two weeks after SCI (n = 5/group). (A1-C1) GFAP, (A2-C2) pEFGR, and (A3-C3) co-localization of pEGFR and GFAP. Insets in panels (B1-C3) are shown at high magnification in panels (D1-E3) , respectively. Scale bars = 500 μm in (C3) (applies to A1-C3 ); scale bars = 50 μm in (E3) (applies to D1-E3 ). Representative Western blots of pEGFR and EGFR expression (n = 3/group), and β-actin was a loading control (F) . Semi-quantitative measurements were obtained by normalizing to β-actin (G) . * P

    Journal: Journal of Neuroinflammation

    Article Title: Epidermal growth factor receptor inhibitor ameliorates excessive astrogliosis and improves the regeneration microenvironment and functional recovery in adult rats following spinal cord injury

    doi: 10.1186/1742-2094-11-71

    Figure Lengend Snippet: PD168393 inhibited epidermal growth factor receptor (EGFR) phosphorylation of astrocytes after spinal cord injury (SCI) in rats. Spinal cord sections were immunostained for phosphorylated EGFR (pEGFR) and glial fibrillary acid protein (GFAP) at two weeks after SCI (n = 5/group). (A1-C1) GFAP, (A2-C2) pEFGR, and (A3-C3) co-localization of pEGFR and GFAP. Insets in panels (B1-C3) are shown at high magnification in panels (D1-E3) , respectively. Scale bars = 500 μm in (C3) (applies to A1-C3 ); scale bars = 50 μm in (E3) (applies to D1-E3 ). Representative Western blots of pEGFR and EGFR expression (n = 3/group), and β-actin was a loading control (F) . Semi-quantitative measurements were obtained by normalizing to β-actin (G) . * P

    Article Snippet: For immunofluorescence double labeling, sections were treated respectively with a combination of two primary antibodies: mouse anti-GFAP (1:300; Sigma, St. Louis, MO, USA) and rabbit anti-EGFR (1:200; Sigma, St. Louis, MO, USA), mouse anti-GFAP (1:300; Sigma, St. Louis, MO, USA) and rabbit anti-pEGFR (1:200; Sigma, St. Louis, MO, USA), rabbit anti-GFAP (1:300; Sigma, St. Louis, MO, USA) and mouse anti-CSPGs (1:200; Sigma, St. Louis, MO, USA) overnight at 4°C.

    Techniques: Western Blot, Expressing

    PD168393 inhibited EGFR phosphorylation of astrocytes in an in vitro scratch wound model. Cultured astrocytes were immunostained for pEGFR, GFAP and DAPI in control (A1-A4) , injury, (B1-B4) and PD168393 (40 μM) treatment groups (C1-C4) at 24 hours after injury (n = 5 in each group). (A1-C1) immunolabeling for pEFGR, (A2-C2) , GFAP, (A3-C3) DAPI and (A4-C4) for co-localization of pEGFR, GFAP and DAPI. Scale bars = 100 μm. Representative Western blots of pEGFR and GFAP expression are shown (n = 3/group) (D) . Semiquantitative analysis of pEGFR as a ratio of EGFR loading and semiquantitative measurements of GFAP were obtained by normalization to β-actin (E) . * P

    Journal: Journal of Neuroinflammation

    Article Title: Epidermal growth factor receptor inhibitor ameliorates excessive astrogliosis and improves the regeneration microenvironment and functional recovery in adult rats following spinal cord injury

    doi: 10.1186/1742-2094-11-71

    Figure Lengend Snippet: PD168393 inhibited EGFR phosphorylation of astrocytes in an in vitro scratch wound model. Cultured astrocytes were immunostained for pEGFR, GFAP and DAPI in control (A1-A4) , injury, (B1-B4) and PD168393 (40 μM) treatment groups (C1-C4) at 24 hours after injury (n = 5 in each group). (A1-C1) immunolabeling for pEFGR, (A2-C2) , GFAP, (A3-C3) DAPI and (A4-C4) for co-localization of pEGFR, GFAP and DAPI. Scale bars = 100 μm. Representative Western blots of pEGFR and GFAP expression are shown (n = 3/group) (D) . Semiquantitative analysis of pEGFR as a ratio of EGFR loading and semiquantitative measurements of GFAP were obtained by normalization to β-actin (E) . * P

    Article Snippet: For immunofluorescence double labeling, sections were treated respectively with a combination of two primary antibodies: mouse anti-GFAP (1:300; Sigma, St. Louis, MO, USA) and rabbit anti-EGFR (1:200; Sigma, St. Louis, MO, USA), mouse anti-GFAP (1:300; Sigma, St. Louis, MO, USA) and rabbit anti-pEGFR (1:200; Sigma, St. Louis, MO, USA), rabbit anti-GFAP (1:300; Sigma, St. Louis, MO, USA) and mouse anti-CSPGs (1:200; Sigma, St. Louis, MO, USA) overnight at 4°C.

    Techniques: In Vitro, Cell Culture, Immunolabeling, Western Blot, Expressing

    Ser77 is phosphorylated by EGF stimulation. (A) Vero cells were transfected with the indicated mutant palladin. Twenty-four hours later, serum-starved cells were stimulated with EGF for 5 min. Mobility shifts were examined by western blotting. (B) Mobility shifts of Δ44-S197G and Δ102-S197G were examined by western blotting. (C) Amino acid sequence from 45 to 102 is indicated. Mobility shift of the indicated mutant palladin was examined by western blotting. (D) Mobility shift of the indicated mutant palladin was examined by western blotting. (E) Vero cells were transfected with either FL-wt or FL-S77.197G palladin. Twenty-four hours later, cells were stimulated with EGF for 5 min. Cells were lysed and immunoprecipited with anti-Myc antibody and blotted with anti-MPM2 antibody.

    Journal: PLoS ONE

    Article Title: Role of Palladin Phosphorylation by Extracellular Signal-Regulated Kinase in Cell Migration

    doi: 10.1371/journal.pone.0029338

    Figure Lengend Snippet: Ser77 is phosphorylated by EGF stimulation. (A) Vero cells were transfected with the indicated mutant palladin. Twenty-four hours later, serum-starved cells were stimulated with EGF for 5 min. Mobility shifts were examined by western blotting. (B) Mobility shifts of Δ44-S197G and Δ102-S197G were examined by western blotting. (C) Amino acid sequence from 45 to 102 is indicated. Mobility shift of the indicated mutant palladin was examined by western blotting. (D) Mobility shift of the indicated mutant palladin was examined by western blotting. (E) Vero cells were transfected with either FL-wt or FL-S77.197G palladin. Twenty-four hours later, cells were stimulated with EGF for 5 min. Cells were lysed and immunoprecipited with anti-Myc antibody and blotted with anti-MPM2 antibody.

    Article Snippet: To confirm the phosphorylation of serine or threonine residues of palladin, we used an anti-MPM2 antibody that recognizes a phosphorylated epitope (Ser/Thr)-Pro.

    Techniques: Transfection, Mutagenesis, Western Blot, Sequencing, Mobility Shift