anti-egfr Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher anti egfr
    <t>EGFR</t> is localized to the plasma membrane in breast cancer cell lines For each cell line, two hundred thousand cells were plated onto coverslips and cultured in growth medium for 48 h. Cells were fixed, permeabilized, and blocked with 20% goat serum. EGFR was detected with <t>Alexa-fluor</t> 488 labeled EGFR antibody (green) and nuclei were identified with DAPI (blue). Imaging was performed using Zeiss Axioplan2 apotome microscope fitted with a 63X 1.25 oil immersion lens at the Microscopy and Imaging Resources Laboratory (Wayne State University, Detroit, MI). Arrows indicate patchy EGFR staining. Scale bars represent a distance of 50 μm. Images are representative of at least three independent experiments.
    Anti Egfr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 251 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti egfr/product/Thermo Fisher
    Average 99 stars, based on 251 article reviews
    Price from $9.99 to $1999.99
    anti egfr - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore anti egfr
    Resistance to Th1 <t>cytokine/trastuzumab-mediated</t> class I restoration and HER2 369-377 -CD8 + T-cell targeting of HER2-expressing cancers by EGF/Heregulin is rescued with inhibition of <t>EGFR</t> 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
    Anti Egfr, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 357 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti egfr/product/Millipore
    Average 99 stars, based on 357 article reviews
    Price from $9.99 to $1999.99
    anti egfr - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    93
    Cell Signaling Technology Inc anti egfr
    Proposed signaling amplifying mechanism in HCT-116 cells exposed to hyperthermia. At 37 °C, mutant KRAS supports ERK signaling, which in turn augments the already deregulated by a mutation WNT/beta-catenin activity. Signaling through <t>HGFR,</t> a transcriptional target of WNT/beta-catenin activity, feeds back positively the ERK and WNT pathways. At 42 °C, hyperthermia-induced EGF and WNT ligands allow for a switch from HGFR to <t>EGFR</t> signaling, and to increasing in time signaling amplifying mechanism.
    Anti Egfr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1912 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti egfr/product/Cell Signaling Technology Inc
    Average 93 stars, based on 1912 article reviews
    Price from $9.99 to $1999.99
    anti egfr - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    94
    Santa Cruz Biotechnology anti egfr
    Analysis of the effect of the cellular content on EGF signaling dynamics. A . Experimental validation of relative expression level of model species. Upper right panel: predicted parameter value regarding the initial concentration of model species. Error bars represent standard deviations of 1,000 samples from an ensemble of the WT and Y992F models. Left and lower right panel: western blot analysis of relative concentration of model species. Unstimulated cell lysates of the WT and Y992F cells were dissolved by <t>SDS-PAGE</t> and probed using <t>anti-EGFR,</t> anti-Cbl, anti-Cbl-b, anti-Grb2, anti-Src, anti-Shp2, anti-Shc, anti-Erk1/2, and anti-β-tubulin as a loading control. Quantitated band intensities were normalized to the values for WT. With regard to Shc and Erk proteins, the intensities of three bands of Shc isoforms and two bands of Erk1 and Erk2 were combined, respectively, for the calculation of the relative protein amount. Error bars represent standard deviations of triplicate samples. *P
    Anti Egfr, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1523 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti egfr/product/Santa Cruz Biotechnology
    Average 94 stars, based on 1523 article reviews
    Price from $9.99 to $1999.99
    anti egfr - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    99
    Abcam anti egfr
    Expression of thymidylate synthase (TS) and related <t>EGFR</t> signaling pathway proteins in tumor xenografts after the administration of different treatments ( n = 6 mice/group). Groups: control; Ico, icotinib; Ico‐Pem, sequential Ico followed by Pem; Ico + Pem, concurrent Ico and Pem; Pem, pemetrexed; Pem‐Ico, sequential Pem followed by Ico. ( a ) The effects of different combinations of Ico and Pem on TS expression and EGFR, <t>AKT,</t> and MAPK phosphorylation in tumor tissues was detected by Western blotting. The relative ( b ) TS ( c ) phospho‐EGFR, ( d ) phospho‐AKT, and ( e ) phospho‐MAPK expression levels. Data are shown as the mean ± standard deviation of triplicate measurements. P
    Anti Egfr, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 488 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti egfr/product/Abcam
    Average 99 stars, based on 488 article reviews
    Price from $9.99 to $1999.99
    anti egfr - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    92
    Cell Signaling Technology Inc anti phospho egfr
    Both <t>PI3K/Akt</t> and MAPK signaling pathways are essential in lung cancer cells harboring wild-type <t>EGFR</t> ( A ) PI3K inhibition suppressed proliferation of lung cancer cells harboring mutant or wild-type EGFR . A549 and PC-9 cells were incubated in the presence of various concentrations of GSK2126458 or PF04691502. Cell extracts were prepared after 1 h of treatment and immunoblotted with the indicated antibodies (upper panel). Cell growth was measured by MTT assay after 72 h (lower panel). ( B ) MEK inhibition suppressed proliferation of lung cancer cells harboring mutant or wild-type EGFR . A549 and PC-9 cells were incubated in the presence of various concentrations of AZD-8330 or TAK-733. Cell extracts were prepared after 1 h of treatment and immunoblotted with the indicated antibodies (upper panel). Cell growth was measured by MTT assay after 72 h (lower panel).
    Anti Phospho Egfr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 433 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti phospho egfr/product/Cell Signaling Technology Inc
    Average 92 stars, based on 433 article reviews
    Price from $9.99 to $1999.99
    anti phospho egfr - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    91
    Cell Signaling Technology Inc rabbit anti egfr
    ADAM8 preferentially localizes to anchoring columns of placental villi. ( A ) Representative immunofluorescence, immunohistochemistry and RNAScope microscopy images of serially sectioned first trimester placental villi (6–11 weeks gestation; n = 5) and decidua (10–12 weeks gestation; n = 4) showing ADAM8 mRNA transcript localization (pink punctate signal) within trophoblast subtypes. Proximal and distal column, as well as interstitial EVT subtypes of trophoblasts are identified by immunostaining of cytokeratin (KRT7) and <t>HLA-G.</t> ‘PCT’ indicates proximal column trophoblast; ‘DCT’ indicates distal column trophoblast; ‘VT’ indicates villous trophoblast; ‘SynT’ indicates syncytiotrophoblast; ‘MC’ indicates placenta mesenchymal core; ‘iEVT’ indicates interstitial extravillous trophoblast; ‘GC’ indicates trophoblast giant cell. The perforated white box indicates enlarged inset image. Black arrowheads denote ADAM8 signal. Bars = 100 μm. ( B ) Fluorescence activated cell-sorting (FACS) plots demonstrate the trophoblast isolation strategy used to purify mesencymal core cells (MC), distal column trophoblasts (DCT) and villous/proximal column trophoblasts (VT). Live cells, depleted of CD31 + (endothelial) and CD45 + (immune) cells using immuno-magnetic beads, were positively gated by 7AAD exclusion. Cells were further segregated by excluding CD45 + immune cells and by cell surface labeling of HLA-G and CD49f. Cell subtype proportions are indicated within each gated population (percent of cells within FACS plot). ( C ) ADAM8 mRNA levels in FACS-purified MCs, VTs and DCTs. Trophoblast subtype purity was assessed by qPCR analysis targeting the pan-trophoblast marker KRT7 , the EVT-marker HLA-G and the mesenchymal lineage marker VIM . GAPDH was used for normalization. Results are presented as mean ± SD in bar graphs from four distinct placental villi specimens ( n = 4); results were analyzed by one-way ANOVA and Dunn’s multiple comparisons test. ( D ) Immunoblot showing protein levels of pro- and active-ADAM8, HLA-G, and <t>EGFR</t> in MC, VT and DCT isolated from placental villi using immuno-magnetic beads. Molecular weights (kDa) are shown to the left and β-actin indicates loading control.
    Rabbit Anti Egfr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 377 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti egfr/product/Cell Signaling Technology Inc
    Average 91 stars, based on 377 article reviews
    Price from $9.99 to $1999.99
    rabbit anti egfr - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    99
    Millipore anti egfr antibodies
    <t>TGF-α-mediated</t> internalization of <t>EGFR</t> and CD81. (A) Huh-7.5 cells were treated with 5 nM TGF-α in the presence or absence of 5 μM erlotinib for 15 min at 37°C and fixed with paraformaldehyde, and immunofluorescence staining
    Anti Egfr Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti egfr antibodies/product/Millipore
    Average 99 stars, based on 52 article reviews
    Price from $9.99 to $1999.99
    anti egfr antibodies - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    egfr  (Abcam)
    99
    Abcam egfr
    Src Family Kinases mediate nEGFR translocation in TNBC (A) Constitutively active Src (caSrc) enhances nEGFR translocation in TNBC cell lines . Cells were transfected with caSrc or an empty vector control for 48 hr prior to stimulation with EGF (5 nM, 45 min) to induce nEGFR translocation. Non-nuclear and nuclear proteins were harvested. nEGFR expression was quantitated using ImageJ software (B) A negative regulator of Src, src-like adaptor protein (SLAP), blocks nEGFR translocation in TNBC cell lines. Cells were transfected with SLAP-FLAG or an empty vector control for 48 hr prior to harvesting non-nuclear and nuclear proteins. nEGFR expression was analyzed. (C) SLAP can interact with <t>EGFR</t> and decrease EGFR activation at Tyrosine 1101. Cells were transfected with SLAP-FLAG or an empty vector control for 48 hr prior to harvesting whole cell lysate. 250 ug of cell lysate was immunoprecipitated with an anti-SLAP antibody. The same lysate was subjected to immunoblot analysis for activation of EGFR at Tyrosine 1101. <t>pEGFR-Y1101</t> activity was quantitated using ImageJ software. Inset 1: EGFR mutated at Tyrosine 1101 is deficient in nuclear localization. Vector, EGFR-WT, and EGFR-Y1101F were transfected into CHOK1 cells for 48 hr prior to stimulation with EGF (5 nM, 45 min). Non-nuclear and nuclear proteins were harvested, and nEGFR expression was analyzed.
    Egfr, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 910 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egfr/product/Abcam
    Average 99 stars, based on 910 article reviews
    Price from $9.99 to $1999.99
    egfr - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    91
    Santa Cruz Biotechnology rabbit anti egfr
    Immunohistochemical analysis of SW620CE2 cells growing in the cecum of nude mice. Double immunofluorescence staining was used for CD31/PECAM-1 and <t>EGFR,</t> <t>pEGFR,</t> VEGFR2, or pVEGFR2. (A) The SW620CE2 cells expressed TGF-α and VEGF but not EGFR or VEGFR. The KM12C cells expressed TGF-α and EGFR (green), and the SKOV3ip1 cells (growing in the peritoneal cavity) expressed VEGF and VEGFR2 (green). In all tumors, the endothelial cells expressed EGFR and VEGFR2 (yellow). (B) Colocalization for VEGFR1 and CD31 (endothelial cells) and F4/80 (macrophages) in SW620CE2 cecal tumors. (C) Expression of HER2 (green) in SKOV3ipl but not SW620CE2 tumors.
    Rabbit Anti Egfr, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 303 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti egfr/product/Santa Cruz Biotechnology
    Average 91 stars, based on 303 article reviews
    Price from $9.99 to $1999.99
    rabbit anti egfr - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    89
    Affibody anti egfr affibody
    Kinase-mediated ligand-free dimers adopt two ECM dimer architectures. a FLImP distribution (grey) of DIII–DIII separations between <t>CF640R-Affibody</t> molecules bound to wtEGFR on CHO cells treated with 1 μM erlotinib, compiled from 29 FLImP measurements (CI ≤ 6 nm), decomposed into a sum of four components. The concentration of CF640R-Affibody was 4 nM. b Number of measurements consistent with the mean distances resolved in the FLImP distribution of wtEGFR (Fig. 2b ) (associated FLImP distributions in Supplementary Fig. 13 ). Errors were assesed with bootstrap-resampling 12 . c wtEGFR and <t>IIIV/KKRE-EGFR</t> phosphorylation in Y1173 on CHO cells treated or untreated with 10 mM MβCD. Box plots show inclusive median as a line, 25th and 75th quartile as edges, calculated on n = 3 repeats (representative western blots in Supplementary Fig. 14 ). d As a treated with 10 mM MβCD, from 20 FLImP measurements (CI ≤ 7 nm). e Pairwise particle colocalisation fraction from two-colour SPT on live cells at 37 °C. f Duration of pairwise interactions ( τ ON ) in e . Horizontal spreads separate data points (~5000) within each condition. g Crystal structure of tethered wtEGFR (PDB ID 1NQL 5 ) highlighting the location of I545K, I556K, I562R, and V592E (yellow). Colours: DI (green), DII (red), DIII (blue), DIV (grey), EGF (orange). h Head-to-head dimer highlighting the residues mutated in the IIIV/KKRE mutant (yellow). i As a but for the IIIV/KKRE-EGFR mutant from 22 FLImP measurements with CI ≤ 7.5 nm. j FRET-DOCA from DI (blue) and DIII (red) to the membrane for wtEGFR + erlotinib, and L680N-EGFR, derived from measurements in Supplementary Fig. 10 . FRET probes as in Fig. 4e . k Ratio between CF640R-9G8-NB and Alexa 488-EgB4-NB binding after chemical fixation. wtEGFR, blue; wtEGFR + erlotinib, green; L680N-EGFR, red. Line, median; box edges, 25th and 75th quartile, crosses 5th and 95th quartile, calculated over 30 repeats. Example images and analysis are in Supplementary Fig. 15 . l As a but for L680N-EGFR-expressing cells, from 20 FLImP measurements (CI ≤ 6 nm). Lower resolution (8 nm) versions of a , d , and l with ~2-fold more FLImP measurements show the profile of the distributions is conserved (Supplementary Fig. 16 )
    Anti Egfr Affibody, supplied by Affibody, used in various techniques. Bioz Stars score: 89/100, based on 231 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti egfr affibody/product/Affibody
    Average 89 stars, based on 231 article reviews
    Price from $9.99 to $1999.99
    anti egfr affibody - by Bioz Stars, 2020-09
    89/100 stars
      Buy from Supplier

    93
    Becton Dickinson anti egfr
    EGFRvIII–stimulated GBP1 expression is <t>p38</t> MAPK dependent A. After 24 h of serum starvation, U87-EGFRvIII cells were treated with DMSO (−), 10 μM of the <t>EGFR</t> tyrosine kinase inhibitor AG1478 (AG), or 20μM of the p38 inhibitor SB203580 (SB) for an additional 24 h before Western blot analysis. B. U87-EGFRvIII cells were transfected with the indicated concentration of p38 siRNA (si-p38) or control siRNA (si-Luc) for 24 h and then serum starved for 24 h followed by Western blot analysis. The p38 siRNAs were described previously [ 10 ]. C. U87-EGFRvIII cells were transfected with pGL3-237 and pRL-TK for 24 h and then serum starved for 24 h. The starved cells were pretreated with DMSO or 20 μM SB203580 for an additional 24 h before reporter assay. This result is expressed as the mean of three independent experiments ± SD. *, P
    Anti Egfr, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 264 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti egfr/product/Becton Dickinson
    Average 93 stars, based on 264 article reviews
    Price from $9.99 to $1999.99
    anti egfr - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    89
    Santa Cruz Biotechnology rabbit polyclonal anti egfr
    Proteasome-dependent degradation of Fhit in 293 cells. ( A ) The 293 cells were transiently transfected with empty vector (lane 1), FHIT (lane 2), or FHIT and SRC (lanes 3 and 4). The sample in lane 4 was from cells pretreated with 15 μM lactacystin. ( B ) The 293 cells were cotransfected with FHIT and <t>EGFR</t> and stimulated with 100 ng/ml of EGF for the indicated time. Cells were pretreated (lanes 7–12) or not (lanes 1–6) with 15 μM lactacystin. Western blot analyses of total lysates were carried out by using Fhit <t>polyclonal</t> antibody (Fhit), a phospho-Fhit polyclonal antibody (P-Fhit), a mAb to phosphotyrosine (PY), a mAb to Src (Src) and a mAb to actin (Actin).
    Rabbit Polyclonal Anti Egfr, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti egfr/product/Santa Cruz Biotechnology
    Average 89 stars, based on 110 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal anti egfr - by Bioz Stars, 2020-09
    89/100 stars
      Buy from Supplier

    Image Search Results


    EGFR is localized to the plasma membrane in breast cancer cell lines For each cell line, two hundred thousand cells were plated onto coverslips and cultured in growth medium for 48 h. Cells were fixed, permeabilized, and blocked with 20% goat serum. EGFR was detected with Alexa-fluor 488 labeled EGFR antibody (green) and nuclei were identified with DAPI (blue). Imaging was performed using Zeiss Axioplan2 apotome microscope fitted with a 63X 1.25 oil immersion lens at the Microscopy and Imaging Resources Laboratory (Wayne State University, Detroit, MI). Arrows indicate patchy EGFR staining. Scale bars represent a distance of 50 μm. Images are representative of at least three independent experiments.

    Journal: Journal of Cellular Physiology

    Article Title: Lipid raft localization of EGFR alters the response of cancer cells to the EGFR tyrosine kinase inhibitor gefitinib

    doi: 10.1002/jcp.22570

    Figure Lengend Snippet: EGFR is localized to the plasma membrane in breast cancer cell lines For each cell line, two hundred thousand cells were plated onto coverslips and cultured in growth medium for 48 h. Cells were fixed, permeabilized, and blocked with 20% goat serum. EGFR was detected with Alexa-fluor 488 labeled EGFR antibody (green) and nuclei were identified with DAPI (blue). Imaging was performed using Zeiss Axioplan2 apotome microscope fitted with a 63X 1.25 oil immersion lens at the Microscopy and Imaging Resources Laboratory (Wayne State University, Detroit, MI). Arrows indicate patchy EGFR staining. Scale bars represent a distance of 50 μm. Images are representative of at least three independent experiments.

    Article Snippet: Anti-EGFR (mab108) was labeled with Alexa-fluor-488 (Invitrogen, Carlsbad, CA).

    Techniques: Cell Culture, Labeling, Imaging, Microscopy, Staining

    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

    Journal: Cancer immunology research

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

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

    Figure Lengend 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

    Article Snippet: HER2-expressing cells were treated with the following, either alone or in designated combinations: rhTNFα, rhIFNγ (BD Biosciences), trastuzumab (Genentech), lapatinib (Santa Cruz Biotechnology); rh-EGF (BD Biosciences), rh-Heregulin (Sigma-Aldrich); neutralizing anti-EGFR (LA1) and/or anti-HER3 (H3.105.5) antibodies, or IgG1 isotype control antibody (all Millipore); and neutralizing anti-PD-1 (MIH1 ( )) or IgG1 isotype control (eBiosciences).

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

    Proposed signaling amplifying mechanism in HCT-116 cells exposed to hyperthermia. At 37 °C, mutant KRAS supports ERK signaling, which in turn augments the already deregulated by a mutation WNT/beta-catenin activity. Signaling through HGFR, a transcriptional target of WNT/beta-catenin activity, feeds back positively the ERK and WNT pathways. At 42 °C, hyperthermia-induced EGF and WNT ligands allow for a switch from HGFR to EGFR signaling, and to increasing in time signaling amplifying mechanism.

    Journal: Cancers

    Article Title: In Hyperthermia Increased ERK and WNT Signaling Suppress Colorectal Cancer Cell Growth

    doi: 10.3390/cancers8050049

    Figure Lengend Snippet: Proposed signaling amplifying mechanism in HCT-116 cells exposed to hyperthermia. At 37 °C, mutant KRAS supports ERK signaling, which in turn augments the already deregulated by a mutation WNT/beta-catenin activity. Signaling through HGFR, a transcriptional target of WNT/beta-catenin activity, feeds back positively the ERK and WNT pathways. At 42 °C, hyperthermia-induced EGF and WNT ligands allow for a switch from HGFR to EGFR signaling, and to increasing in time signaling amplifying mechanism.

    Article Snippet: The following antibodies were used: anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#4370, Cell Signaling Technology, Beverly, MA, USA), anti-phospho-STAT3 (Tyr705) (#9145, Cell Signaling Technology), anti-Ser473-phosphorylated AKT (sc-7985, Santa Cruz Biotechnology, anti-AKT (sc-8312, Santa Cruz Biotechnology), anti-beta-ACTIN (A5441, Sigma-Aldrich), anti-ERK1/2 (#9102, Cell Signaling Technology), anti-SOS1 (#07-337, Millipore, Billerica, MA, USA), anti-STAT3 (#9139, Cell Signaling Technology), anti-pcJUN, anti-c-JUN (sc-16312-R and sc-45, Santa Cruz Biotechnology), anti-pEGFR (pTyr1068, #3777, Cell Signaling Technology), anti-pHGFR (pTyr1234/1235, #3077, Cell Signaling Technology), anti-EGFR (#4267, Cell Signaling Technology) and anti-HGFR (#3127, Cell Signaling Technology), anti-beta-catenin (sc-53483, Santa Cruz Biotechnology) and anti-active beta-catenin antibody that recognizes the protein only when dephosphorylated on Ser37 or Thr41 (#05-665, Millipore).

    Techniques: Mutagenesis, Activity Assay

    EGFR expression in Kupffer cells/liver macrophages promotes HCC development ( a, b ) Numbers of F4/80 + cells in tumors of mice (left, EGFR f/f : n=66 (4 mice), EGFR Δhep : n=54 (4 mice), EGFR f/f : n=37 (3 mice), EGFR ΔMx : n=36 HPF (4 mice)) and CCL2 serum levels in HCC mice (right, EGFR f/f : n=4, EGFR Δhep : n=7, EGFR f/f : n=4 and EGFR ΔMx : n=6 mice). ( c ) Representative PCR showing EGFR deletion in isolated hepatocytes (Hep) and Kupffer cells (KC) of control (f/Δ) and EGFR Δhep/Δmac mice. flox = not deleted (1.1kb) and Δ = deleted EGFR (0.5kb). ( d ) Representative livers (top, scale bar: 1cm) and H E stainings of sections of indicated genotypes (bottom, scale bars: 1mm) 63 weeks after tumor initiation. Dotted lines mark tumor nodules. Note: Tumors of EGFR f/f mice are bigger than in Fig. 2c, d , because the tumors were analyzed 27 weeks later due to a change in the genetic background of the mice. ( e ) Tumor mass in livers of EGFR f/f (n=10), EGFR Δhep (n=5), EGFR Δhep/Δmac (n=5) and EGFR Δmac (n=4) mice. Data ( a, b ) represent mean±s.e.m. Data ( e ) represent mean ±s.d. Student‘s t -test for independent samples and unequal variances was used to assess statistical significance (*p

    Journal: Nature cell biology

    Article Title: EGFR has a tumor-promoting role in liver macrophages during hepatocellular carcinoma formation

    doi: 10.1038/ncb3031

    Figure Lengend Snippet: EGFR expression in Kupffer cells/liver macrophages promotes HCC development ( a, b ) Numbers of F4/80 + cells in tumors of mice (left, EGFR f/f : n=66 (4 mice), EGFR Δhep : n=54 (4 mice), EGFR f/f : n=37 (3 mice), EGFR ΔMx : n=36 HPF (4 mice)) and CCL2 serum levels in HCC mice (right, EGFR f/f : n=4, EGFR Δhep : n=7, EGFR f/f : n=4 and EGFR ΔMx : n=6 mice). ( c ) Representative PCR showing EGFR deletion in isolated hepatocytes (Hep) and Kupffer cells (KC) of control (f/Δ) and EGFR Δhep/Δmac mice. flox = not deleted (1.1kb) and Δ = deleted EGFR (0.5kb). ( d ) Representative livers (top, scale bar: 1cm) and H E stainings of sections of indicated genotypes (bottom, scale bars: 1mm) 63 weeks after tumor initiation. Dotted lines mark tumor nodules. Note: Tumors of EGFR f/f mice are bigger than in Fig. 2c, d , because the tumors were analyzed 27 weeks later due to a change in the genetic background of the mice. ( e ) Tumor mass in livers of EGFR f/f (n=10), EGFR Δhep (n=5), EGFR Δhep/Δmac (n=5) and EGFR Δmac (n=4) mice. Data ( a, b ) represent mean±s.e.m. Data ( e ) represent mean ±s.d. Student‘s t -test for independent samples and unequal variances was used to assess statistical significance (*p

    Article Snippet: For immunohistochemistry and immunofluorescent stainings, antibodies against the following antigens were used: EGFR (CST #4267; clone D38B1; 1/50), F4/80 (Serotec MCA497R, clone CI:A3-1; 1/100 and eBioscience 14-4801; clone BM8; 1/100), Ki67 (Novo Castra NCL-Ki67p; 1/1000), CD68 (abcam ab955; clone KP1; 1/200), HMGB1 (CST #3935; 1/100), active Caspase 3 (R & D Systems AF835; 1/2000).

    Techniques: Expressing, Mouse Assay, Polymerase Chain Reaction, Isolation

    EGFR expression is induced in activated Kupffer cells/liver macrophages under pathological conditions ( a-b ) Representative immunofluorescent confocal image showing co-staining for F4/80 and EGFR in cultured Kupffer cells/liver macrophages isolated from ( a ) EGFR f/f and ( b ) EGFR Δmac livers and stimulated with IL-1β for 24 h. Cultures contained ≥ 98% Kupffer cells/liver macrophages as confirmed by F4/80 staining. Scale bar: 50μm. ( c ) Representative Western Blot showing EGFR expression in isolated hepatocytes and Kupffer cells of EGFR f/f , EGFR ΔMx , EGFR Δhep and EGFR Δmac mice. ( d-e ) Representative immunofluorescent confocal images showing F4/80 and EGFR expression in liver sections of ( d ) untreated and ( e ) DEN treated (5 days) EGFR f/f mice. White arrows indicate EGFR-positive Kupffer cells. Scale bar: 50μm. ( f-g ) Mean fluorescence intensity (mean FI) showing EGFR expression levels (Alexa 488, green) in ( f ) liver macrophages ( EGFR f/f untreated: EGFR negative (n=9), EGFR positive (n=10); EGFR f/f 5 days after DEN: EGFR negative (n=4), EGFR positive (n=26) and ( g ) hepatocytes ( EGFR f/f untreated (n=12), EGFR f/f 5 days after DEN (n=13). Analysis of stainings shown in ( d ) and ( e ). Two pooled independent experiments for ( f ) and ( g ). ( h-k ) Representative anti-EGFR ( h, i ) and anti-F4/80 ( j, k ) staining performed on serial sections of control ( h, j ) and EGFR Δhep/Δmac ( i, k ) HCC showing EGFR expression in tumor cells and co-expression of EGFR and F4/80 in Kupffer cells/liver macrophages of EGFR f/f HCC and no EGFR expression in EGFR Δhep/Δmac tumors. Scale bar: 50μm. ( a-b, d-e ) Nuclei (DAPI, blue), EGFR (Alexa 488, green) and F4/80 (Alexa 594, red), merge (bottom right). Data ( f-g ) represent mean±s.d. Student‘s t -test for independent samples and unequal variances was used to assess statistical significance (*p

    Journal: Nature cell biology

    Article Title: EGFR has a tumor-promoting role in liver macrophages during hepatocellular carcinoma formation

    doi: 10.1038/ncb3031

    Figure Lengend Snippet: EGFR expression is induced in activated Kupffer cells/liver macrophages under pathological conditions ( a-b ) Representative immunofluorescent confocal image showing co-staining for F4/80 and EGFR in cultured Kupffer cells/liver macrophages isolated from ( a ) EGFR f/f and ( b ) EGFR Δmac livers and stimulated with IL-1β for 24 h. Cultures contained ≥ 98% Kupffer cells/liver macrophages as confirmed by F4/80 staining. Scale bar: 50μm. ( c ) Representative Western Blot showing EGFR expression in isolated hepatocytes and Kupffer cells of EGFR f/f , EGFR ΔMx , EGFR Δhep and EGFR Δmac mice. ( d-e ) Representative immunofluorescent confocal images showing F4/80 and EGFR expression in liver sections of ( d ) untreated and ( e ) DEN treated (5 days) EGFR f/f mice. White arrows indicate EGFR-positive Kupffer cells. Scale bar: 50μm. ( f-g ) Mean fluorescence intensity (mean FI) showing EGFR expression levels (Alexa 488, green) in ( f ) liver macrophages ( EGFR f/f untreated: EGFR negative (n=9), EGFR positive (n=10); EGFR f/f 5 days after DEN: EGFR negative (n=4), EGFR positive (n=26) and ( g ) hepatocytes ( EGFR f/f untreated (n=12), EGFR f/f 5 days after DEN (n=13). Analysis of stainings shown in ( d ) and ( e ). Two pooled independent experiments for ( f ) and ( g ). ( h-k ) Representative anti-EGFR ( h, i ) and anti-F4/80 ( j, k ) staining performed on serial sections of control ( h, j ) and EGFR Δhep/Δmac ( i, k ) HCC showing EGFR expression in tumor cells and co-expression of EGFR and F4/80 in Kupffer cells/liver macrophages of EGFR f/f HCC and no EGFR expression in EGFR Δhep/Δmac tumors. Scale bar: 50μm. ( a-b, d-e ) Nuclei (DAPI, blue), EGFR (Alexa 488, green) and F4/80 (Alexa 594, red), merge (bottom right). Data ( f-g ) represent mean±s.d. Student‘s t -test for independent samples and unequal variances was used to assess statistical significance (*p

    Article Snippet: For immunohistochemistry and immunofluorescent stainings, antibodies against the following antigens were used: EGFR (CST #4267; clone D38B1; 1/50), F4/80 (Serotec MCA497R, clone CI:A3-1; 1/100 and eBioscience 14-4801; clone BM8; 1/100), Ki67 (Novo Castra NCL-Ki67p; 1/1000), CD68 (abcam ab955; clone KP1; 1/200), HMGB1 (CST #3935; 1/100), active Caspase 3 (R & D Systems AF835; 1/2000).

    Techniques: Expressing, Staining, Cell Culture, Isolation, Western Blot, Mouse Assay, Fluorescence

    Shikonin inhibited EGFR/PI3K/AKT signal pathway. Ec109 and EC9706 cells were treated with shikonin(0,2,4μg/ml) for 24h and the total protein were extracted, then the expression of EGFR, PI3K, AKT, P-AKT, mTOR, PKM2 and HIF1ɑ were examined by Western blot. The results showed that shikonin decreased the expression of EGFR, PI3K, AKT, P-AKT, mTOR, PKM2 and HIF1ɑ.

    Journal: Journal of Cancer

    Article Title: Efficacy of Shikonin against Esophageal Cancer Cells and its possible mechanisms in vitro and in vivo

    doi: 10.7150/jca.21224

    Figure Lengend Snippet: Shikonin inhibited EGFR/PI3K/AKT signal pathway. Ec109 and EC9706 cells were treated with shikonin(0,2,4μg/ml) for 24h and the total protein were extracted, then the expression of EGFR, PI3K, AKT, P-AKT, mTOR, PKM2 and HIF1ɑ were examined by Western blot. The results showed that shikonin decreased the expression of EGFR, PI3K, AKT, P-AKT, mTOR, PKM2 and HIF1ɑ.

    Article Snippet: Antibodies against EGFR, PI3K, AKT, P-AKT and mTOR were purchased from Cell signaling Technology (USA).

    Techniques: Expressing, Western Blot

    Validation of specific peptide binding to EGFR. On confocal microscopy, we found strong binding of ( a ) QRH*-Cy5.5 peptide (red) and ( b ) AF488-labeled anti-EGFR (green) to the surface (arrow) of control HT29 cells (siCL). ( c ) PEH*-Cy5.5 (red) binding is minimal. ( d – f ) The fluorescence intensities are significantly reduced in knockdown of HT29 cells (siEGFR). ( g ) Quantified results for QRH*-Cy5.5 and anti-EGFR show significantly higher intensities for siCL- vs. siEGFR-transfected cells (3.2- and 3.4-fold change, P =0.0021 and 0.0017, respectively), whereas PEH*-Cy5.5 showed a nonsignificant decrease (0.87 fold-change, P =0.57). Differences for siCL vs. siEGFR for QRH*-Cy5.5 and anti-EGFR were significantly greater than those for PEH*-Cy5.5 ( P =0.007 and 0.006, respectively). We fit two-way ANOVA models with the terms for six conditions and two replicate slides on log-transformed data. Measurements were on an average of five randomly chosen cells on two slides for each condition. ( h ) Western blot shows EGFR expression levels. ( i ) On competition, we found a significant difference in binding of QRH*-Cy5.5 to HT29 cells with the addition of unlabeled QRH* and PEH* at the concentrations of 50 μ m and higher. Nonsignficant difference was found at 0 μ m . We fit two-way ANOVA models with the terms for the labeled peptide, concentrations of the unlabeled peptides, and their interactions on log-transformed data. P -values shown here compare the difference in the intensity between unlabeled QRH* and PEH* at each dose with that at 0 μ m . Measurements are on an average of five randomly chosen cells on two slides for each condition. ANOVA, analysis of variance; EGFR, epidermal growth factor receptor.

    Journal: Clinical and Translational Gastroenterology

    Article Title: EGFR Overexpressed in Colonic Neoplasia Can be Detected on Wide-Field Endoscopic Imaging

    doi: 10.1038/ctg.2015.28

    Figure Lengend Snippet: Validation of specific peptide binding to EGFR. On confocal microscopy, we found strong binding of ( a ) QRH*-Cy5.5 peptide (red) and ( b ) AF488-labeled anti-EGFR (green) to the surface (arrow) of control HT29 cells (siCL). ( c ) PEH*-Cy5.5 (red) binding is minimal. ( d – f ) The fluorescence intensities are significantly reduced in knockdown of HT29 cells (siEGFR). ( g ) Quantified results for QRH*-Cy5.5 and anti-EGFR show significantly higher intensities for siCL- vs. siEGFR-transfected cells (3.2- and 3.4-fold change, P =0.0021 and 0.0017, respectively), whereas PEH*-Cy5.5 showed a nonsignificant decrease (0.87 fold-change, P =0.57). Differences for siCL vs. siEGFR for QRH*-Cy5.5 and anti-EGFR were significantly greater than those for PEH*-Cy5.5 ( P =0.007 and 0.006, respectively). We fit two-way ANOVA models with the terms for six conditions and two replicate slides on log-transformed data. Measurements were on an average of five randomly chosen cells on two slides for each condition. ( h ) Western blot shows EGFR expression levels. ( i ) On competition, we found a significant difference in binding of QRH*-Cy5.5 to HT29 cells with the addition of unlabeled QRH* and PEH* at the concentrations of 50 μ m and higher. Nonsignficant difference was found at 0 μ m . We fit two-way ANOVA models with the terms for the labeled peptide, concentrations of the unlabeled peptides, and their interactions on log-transformed data. P -values shown here compare the difference in the intensity between unlabeled QRH* and PEH* at each dose with that at 0 μ m . Measurements are on an average of five randomly chosen cells on two slides for each condition. ANOVA, analysis of variance; EGFR, epidermal growth factor receptor.

    Article Snippet: QRH*-Cy5.5 and anti-EGFR antibody showed significantly higher intensities for siCL-transfected HT29 cells than for those treated with siEGFR, whereas PEH*-Cy5.5 showed a small nonsignificant increase, .

    Techniques: Binding Assay, Confocal Microscopy, Labeling, Fluorescence, Transfection, Transformation Assay, Western Blot, Expressing

    Characterization of EGFR peptide-binding parameters. ( a ) Apparent dissociation constant k d =50 n m , R 2 =0.95 was measured for binding of QRH*-Cy5.5 to HT29 cells. ( b ) Apparent association time constant k =0.406/min (2.46 min) was measured for binding of QRH*-Cy5.5 to HT29 cells. Both results are representative of six independent experiments.

    Journal: Clinical and Translational Gastroenterology

    Article Title: EGFR Overexpressed in Colonic Neoplasia Can be Detected on Wide-Field Endoscopic Imaging

    doi: 10.1038/ctg.2015.28

    Figure Lengend Snippet: Characterization of EGFR peptide-binding parameters. ( a ) Apparent dissociation constant k d =50 n m , R 2 =0.95 was measured for binding of QRH*-Cy5.5 to HT29 cells. ( b ) Apparent association time constant k =0.406/min (2.46 min) was measured for binding of QRH*-Cy5.5 to HT29 cells. Both results are representative of six independent experiments.

    Article Snippet: QRH*-Cy5.5 and anti-EGFR antibody showed significantly higher intensities for siCL-transfected HT29 cells than for those treated with siEGFR, whereas PEH*-Cy5.5 showed a small nonsignificant increase, .

    Techniques: Binding Assay

    Peptide specific for EGFR. ( a ) Chemical structure of QRHKPRE peptide (black) with GGGSK linker (blue) and Cy5.5 fluorophore (red). ( b ) Scrambled peptide PEHKRRQ (control). ( c ) QRH*-Cy5.5 was found on the structural model to bind domain 2 of EGFR (1IVO). ( d ) Fluorescence spectra of Cy5.5-labeled peptides with λ ex =671 nm shows peak emission near 710 nm. AU, arbitrary unit; EGFR, epidermal growth factor receptor.

    Journal: Clinical and Translational Gastroenterology

    Article Title: EGFR Overexpressed in Colonic Neoplasia Can be Detected on Wide-Field Endoscopic Imaging

    doi: 10.1038/ctg.2015.28

    Figure Lengend Snippet: Peptide specific for EGFR. ( a ) Chemical structure of QRHKPRE peptide (black) with GGGSK linker (blue) and Cy5.5 fluorophore (red). ( b ) Scrambled peptide PEHKRRQ (control). ( c ) QRH*-Cy5.5 was found on the structural model to bind domain 2 of EGFR (1IVO). ( d ) Fluorescence spectra of Cy5.5-labeled peptides with λ ex =671 nm shows peak emission near 710 nm. AU, arbitrary unit; EGFR, epidermal growth factor receptor.

    Article Snippet: QRH*-Cy5.5 and anti-EGFR antibody showed significantly higher intensities for siCL-transfected HT29 cells than for those treated with siEGFR, whereas PEH*-Cy5.5 showed a small nonsignificant increase, .

    Techniques: Fluorescence, Labeling

    Binding of EGFR peptide and antibody to human colonic neoplasia. On confocal microscopy, binding of ( a ) QRH*-Cy5.5 peptide (red) co-localizes with that of ( b ) AF488-labeled anti-EGFR antibody (green) on surface of dysplastic colonocytes (arrow), shown in ( c ) merged image, P= 0.71. ( d ) Image contrast can be appreciated at lesion border. Magnified view of boxes in d is shown for ( e ) dysplasia and ( f ) normal. ( g ) Corresponding immunohistochemistry from a shows increased reactivity for EGFR in dysplasia. ( h ) Dysplasia ( n =29) showed significantly higher fluorescence intensities than normal ( n =15) by an average of 19.4-fold, P =1.7 × 10 −9 by two-sample t -test on log-transformed data. ( i ) Receiver operating characteristic curve shows 90% sensitivity and 93% specificity with area under curve (AUC) of 0.94 for distinguishing dysplasia from normal using peptide.

    Journal: Clinical and Translational Gastroenterology

    Article Title: EGFR Overexpressed in Colonic Neoplasia Can be Detected on Wide-Field Endoscopic Imaging

    doi: 10.1038/ctg.2015.28

    Figure Lengend Snippet: Binding of EGFR peptide and antibody to human colonic neoplasia. On confocal microscopy, binding of ( a ) QRH*-Cy5.5 peptide (red) co-localizes with that of ( b ) AF488-labeled anti-EGFR antibody (green) on surface of dysplastic colonocytes (arrow), shown in ( c ) merged image, P= 0.71. ( d ) Image contrast can be appreciated at lesion border. Magnified view of boxes in d is shown for ( e ) dysplasia and ( f ) normal. ( g ) Corresponding immunohistochemistry from a shows increased reactivity for EGFR in dysplasia. ( h ) Dysplasia ( n =29) showed significantly higher fluorescence intensities than normal ( n =15) by an average of 19.4-fold, P =1.7 × 10 −9 by two-sample t -test on log-transformed data. ( i ) Receiver operating characteristic curve shows 90% sensitivity and 93% specificity with area under curve (AUC) of 0.94 for distinguishing dysplasia from normal using peptide.

    Article Snippet: QRH*-Cy5.5 and anti-EGFR antibody showed significantly higher intensities for siCL-transfected HT29 cells than for those treated with siEGFR, whereas PEH*-Cy5.5 showed a small nonsignificant increase, .

    Techniques: Binding Assay, Confocal Microscopy, Labeling, Immunohistochemistry, Fluorescence, Transformation Assay

    HPV(-) cells overexpressing E6/E7 plasmid downregulate miR-133a-3p expression and in turn modulate EGFR and HuR protein expression. (A) UMSCC11A cells were treated with 10% CSE for eight days and tested for miR-133a-3p levels by qRT-PCR. Actin and RPS18 were used as endogenous controls. (B) UMSCC11A cells were treated with 10% CSE for eight days and tested for EGFR and HuR proteins. β-Actin was used as loading control. (C) UMSCC11A cells were transfected with E6/E7 overexpression constructs, identified using semi-quantitative PCR and the DNA was separated by 2% agarose gel electrophoresis. Bands shown with arrowheads indicate the expression of E6 and E7 genes after eight days of incubation. UMSCC47 genomic DNA was used here as a positive control for E6 and E7. * denotes non-specific bands. (D) UMSCC11A cells overexpressing E6/E7 constructs were treated with 10% CSE for eight days, and the miR-133a-3p level was determined between zero and eight days of 10% CSE treatment. *denotes p -value of

    Journal: PLoS ONE

    Article Title: Smoking-induced control of miR-133a-3p alters the expression of EGFR and HuR in HPV-infected oropharyngeal cancer

    doi: 10.1371/journal.pone.0205077

    Figure Lengend Snippet: HPV(-) cells overexpressing E6/E7 plasmid downregulate miR-133a-3p expression and in turn modulate EGFR and HuR protein expression. (A) UMSCC11A cells were treated with 10% CSE for eight days and tested for miR-133a-3p levels by qRT-PCR. Actin and RPS18 were used as endogenous controls. (B) UMSCC11A cells were treated with 10% CSE for eight days and tested for EGFR and HuR proteins. β-Actin was used as loading control. (C) UMSCC11A cells were transfected with E6/E7 overexpression constructs, identified using semi-quantitative PCR and the DNA was separated by 2% agarose gel electrophoresis. Bands shown with arrowheads indicate the expression of E6 and E7 genes after eight days of incubation. UMSCC47 genomic DNA was used here as a positive control for E6 and E7. * denotes non-specific bands. (D) UMSCC11A cells overexpressing E6/E7 constructs were treated with 10% CSE for eight days, and the miR-133a-3p level was determined between zero and eight days of 10% CSE treatment. *denotes p -value of

    Article Snippet: HuR antibody was obtained from Santa Cruz (cat# sc-5261), EGFR antibody obtained from Cell signaling technology (Cat# 2232S), β-Actin monoclonal antibody was purchased from Proteintech (cat # 60008-1-IG).

    Techniques: Plasmid Preparation, Expressing, Quantitative RT-PCR, Transfection, Over Expression, Construct, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Incubation, Positive Control

    Analysis of the effect of the cellular content on EGF signaling dynamics. A . Experimental validation of relative expression level of model species. Upper right panel: predicted parameter value regarding the initial concentration of model species. Error bars represent standard deviations of 1,000 samples from an ensemble of the WT and Y992F models. Left and lower right panel: western blot analysis of relative concentration of model species. Unstimulated cell lysates of the WT and Y992F cells were dissolved by SDS-PAGE and probed using anti-EGFR, anti-Cbl, anti-Cbl-b, anti-Grb2, anti-Src, anti-Shp2, anti-Shc, anti-Erk1/2, and anti-β-tubulin as a loading control. Quantitated band intensities were normalized to the values for WT. With regard to Shc and Erk proteins, the intensities of three bands of Shc isoforms and two bands of Erk1 and Erk2 were combined, respectively, for the calculation of the relative protein amount. Error bars represent standard deviations of triplicate samples. *P

    Journal: PLoS ONE

    Article Title: Phosphoproteomics-Based Modeling Defines the Regulatory Mechanism Underlying Aberrant EGFR Signaling

    doi: 10.1371/journal.pone.0013926

    Figure Lengend Snippet: Analysis of the effect of the cellular content on EGF signaling dynamics. A . Experimental validation of relative expression level of model species. Upper right panel: predicted parameter value regarding the initial concentration of model species. Error bars represent standard deviations of 1,000 samples from an ensemble of the WT and Y992F models. Left and lower right panel: western blot analysis of relative concentration of model species. Unstimulated cell lysates of the WT and Y992F cells were dissolved by SDS-PAGE and probed using anti-EGFR, anti-Cbl, anti-Cbl-b, anti-Grb2, anti-Src, anti-Shp2, anti-Shc, anti-Erk1/2, and anti-β-tubulin as a loading control. Quantitated band intensities were normalized to the values for WT. With regard to Shc and Erk proteins, the intensities of three bands of Shc isoforms and two bands of Erk1 and Erk2 were combined, respectively, for the calculation of the relative protein amount. Error bars represent standard deviations of triplicate samples. *P

    Article Snippet: Extracted protein samples were dissolved by SDS-PAGE probed using anti-EGFR and anti-β-tubulin antibodies as a loading control. (0.66 MB TIF) Click here for additional data file.

    Techniques: Expressing, Concentration Assay, Western Blot, SDS Page

    Recruitment and phosphorylation of EGFR differs in response to stimuli. A: The HCLEs were cultured to confluence and stimulated with control media change (−) over 60 minutes. The EGF (5 minutes) was used as a positive control (+). The pEGFR was detected using a phosphorylated tyrosine (p-tyr) antibody and is normalized to EGFR. B through D: The HCLEs were cultured to confluence and stimulated with EGF (0.5 and 5 nmol/L) or UTP (100 μmol/L) over 60 minutes. Confluent cells were lysed, and EGFR was immunoprecipitated from the lysates. The SDS-PAGE and Western blot analyses of the immunoprecipitation were performed for EGFR, Shc, Src, and Grb2; and probed for p-tyr to determine pEGFR. Images are representative of four experiments. E and F: Densitometric analysis [5.0-nmol/L EGF (black); 0.5-nmol/L EGF (light gray); and 100-μmol/L UTP (dark gray)]. Images are representative of four independent experiments.

    Journal: The American Journal of Pathology

    Article Title: Distinct Activation of Epidermal Growth Factor Receptor by UTP Contributes to Epithelial Cell Wound Repair

    doi: 10.1016/j.ajpath.2010.11.060

    Figure Lengend Snippet: Recruitment and phosphorylation of EGFR differs in response to stimuli. A: The HCLEs were cultured to confluence and stimulated with control media change (−) over 60 minutes. The EGF (5 minutes) was used as a positive control (+). The pEGFR was detected using a phosphorylated tyrosine (p-tyr) antibody and is normalized to EGFR. B through D: The HCLEs were cultured to confluence and stimulated with EGF (0.5 and 5 nmol/L) or UTP (100 μmol/L) over 60 minutes. Confluent cells were lysed, and EGFR was immunoprecipitated from the lysates. The SDS-PAGE and Western blot analyses of the immunoprecipitation were performed for EGFR, Shc, Src, and Grb2; and probed for p-tyr to determine pEGFR. Images are representative of four experiments. E and F: Densitometric analysis [5.0-nmol/L EGF (black); 0.5-nmol/L EGF (light gray); and 100-μmol/L UTP (dark gray)]. Images are representative of four independent experiments.

    Article Snippet: For immunoprecipitation, magnetic beads (Dynabead IgG; Invitrogen) were prepared with antibody directed against EGFR, 40 μL (Santa Cruz Biotechnology, Santa Cruz, CA), according to manufacturer's instructions.

    Techniques: Cell Culture, Positive Control, Immunoprecipitation, SDS Page, Western Blot

    The HCLEs were stimulated with HB-EGF. A: The HCLEs were cultured to confluence and stimulated with HB-EGF (1 nmol/L) over 60 minutes. The EGFR was immunoprecipitated from the lysates, and SDS-PAGE and Western blot analyses of the immunoprecipitation were performed. Antibodies directed against EGFR and Grb2 and an antibody to p-tyr were used. The pEGFR was normalized to EGFR. B: The HCLEs were cultured to confluence, preincubated for 20 minutes with a functional blocking antibody to HB-EGF (40 μg/ml) or control media (−), and stimulated with media change (−), EGF (1 nmol/L), HB-EGF (1 nmol/L), or UTP (100 μmol/L) for 5 minutes. Lysates were extracted, and EGFR was immunoprecipitated. The EGFR and p-tyr antibodies were used, and pEGFR was normalized to EGFR.

    Journal: The American Journal of Pathology

    Article Title: Distinct Activation of Epidermal Growth Factor Receptor by UTP Contributes to Epithelial Cell Wound Repair

    doi: 10.1016/j.ajpath.2010.11.060

    Figure Lengend Snippet: The HCLEs were stimulated with HB-EGF. A: The HCLEs were cultured to confluence and stimulated with HB-EGF (1 nmol/L) over 60 minutes. The EGFR was immunoprecipitated from the lysates, and SDS-PAGE and Western blot analyses of the immunoprecipitation were performed. Antibodies directed against EGFR and Grb2 and an antibody to p-tyr were used. The pEGFR was normalized to EGFR. B: The HCLEs were cultured to confluence, preincubated for 20 minutes with a functional blocking antibody to HB-EGF (40 μg/ml) or control media (−), and stimulated with media change (−), EGF (1 nmol/L), HB-EGF (1 nmol/L), or UTP (100 μmol/L) for 5 minutes. Lysates were extracted, and EGFR was immunoprecipitated. The EGFR and p-tyr antibodies were used, and pEGFR was normalized to EGFR.

    Article Snippet: For immunoprecipitation, magnetic beads (Dynabead IgG; Invitrogen) were prepared with antibody directed against EGFR, 40 μL (Santa Cruz Biotechnology, Santa Cruz, CA), according to manufacturer's instructions.

    Techniques: Cell Culture, Immunoprecipitation, SDS Page, Western Blot, Functional Assay, Blocking Assay

    Injury and UTP recruit specific docking proteins to the EGFR. Comparisons were made in two cell lines and primary epithelial cells. The HCLEs ( A ), primary corneal epithelial cells ( B ), and E1-PAEs ( C ) were stimulated with a control media change (C), injury (W), UTP (25 μmol/L), or EGF (5 nmol/L) for 5 minutes. Cells were lysed, and EGFR was immunoprecipitated from the lysates. SDS-PAGE and Western blot analyses of the immunoprecipitation were performed for EGFR, PLCγ1, Src, Shc, and Grb2. D: Phosphorylation of EGFR was determined after HCLEs were stimulated with media change (control), EGF (0.5 and 5 nmol/L), and UTP (100 μmol/L) at 5 and 60 minutes. SDS-PAGE and Western blot analyses of the immunoprecipitation were performed for EGFR. The pEGFR was detected using a phosphorylated tyrosine antibody. The pEGFR is normalized to EGFR. Data represent a minimum of three independent experiments.

    Journal: The American Journal of Pathology

    Article Title: Distinct Activation of Epidermal Growth Factor Receptor by UTP Contributes to Epithelial Cell Wound Repair

    doi: 10.1016/j.ajpath.2010.11.060

    Figure Lengend Snippet: Injury and UTP recruit specific docking proteins to the EGFR. Comparisons were made in two cell lines and primary epithelial cells. The HCLEs ( A ), primary corneal epithelial cells ( B ), and E1-PAEs ( C ) were stimulated with a control media change (C), injury (W), UTP (25 μmol/L), or EGF (5 nmol/L) for 5 minutes. Cells were lysed, and EGFR was immunoprecipitated from the lysates. SDS-PAGE and Western blot analyses of the immunoprecipitation were performed for EGFR, PLCγ1, Src, Shc, and Grb2. D: Phosphorylation of EGFR was determined after HCLEs were stimulated with media change (control), EGF (0.5 and 5 nmol/L), and UTP (100 μmol/L) at 5 and 60 minutes. SDS-PAGE and Western blot analyses of the immunoprecipitation were performed for EGFR. The pEGFR was detected using a phosphorylated tyrosine antibody. The pEGFR is normalized to EGFR. Data represent a minimum of three independent experiments.

    Article Snippet: For immunoprecipitation, magnetic beads (Dynabead IgG; Invitrogen) were prepared with antibody directed against EGFR, 40 μL (Santa Cruz Biotechnology, Santa Cruz, CA), according to manufacturer's instructions.

    Techniques: Immunoprecipitation, SDS Page, Western Blot

    (A) Confirmation of the expression of intrinsic biomarkers-EGFR, CD44 and TGFβRII in 10 (a) and 15 μg (b) of total protein extracted from cultured MDA-MB-231 by western blot. Total proteins from MDA-MB-231 cells were electrophoresed under same experimental conditions and probed for β-actin, CD44 and TGFβRII proteins using respective primary antibodies followed by corresponding secondary antibodies. EGFR was detected by electrophoresing total proteins in SDS free conditions followed by probing using corresponding primary and secondary antibodies. (B) Cell viability study using Cell Counting Kit-8 on MDA-MB-231 cells exposed to pure AuNP, antibody-bioconjugated (w ab) and non bioconjugated (w/o ab) SERS nanotags (MGITC, Cy5 and Rh6G) after 24 hours.

    Journal: Scientific Reports

    Article Title: Actively Targeted In Vivo Multiplex Detection of Intrinsic Cancer Biomarkers Using Biocompatible SERS Nanotags

    doi: 10.1038/srep04075

    Figure Lengend Snippet: (A) Confirmation of the expression of intrinsic biomarkers-EGFR, CD44 and TGFβRII in 10 (a) and 15 μg (b) of total protein extracted from cultured MDA-MB-231 by western blot. Total proteins from MDA-MB-231 cells were electrophoresed under same experimental conditions and probed for β-actin, CD44 and TGFβRII proteins using respective primary antibodies followed by corresponding secondary antibodies. EGFR was detected by electrophoresing total proteins in SDS free conditions followed by probing using corresponding primary and secondary antibodies. (B) Cell viability study using Cell Counting Kit-8 on MDA-MB-231 cells exposed to pure AuNP, antibody-bioconjugated (w ab) and non bioconjugated (w/o ab) SERS nanotags (MGITC, Cy5 and Rh6G) after 24 hours.

    Article Snippet: For EGFR, 10 μg and 15 μg of the native protein samples were electrophoresed on a 10% Tris gel (with no SDS) with Tris running buffer (with no SDS) and blotted on to a PVDF membrane and subsequently probed with anti-EGFR antibodies (sc120, Santacruz).

    Techniques: Expressing, Cell Culture, Multiple Displacement Amplification, Western Blot, Cell Counting

    EGFR exists in the mitochondria of NSCLC cells and translocates through endocytosis A. Western blotting of the subcellular fractions of CL1–0 and CL1–5 cells. Vinculin and MTCO1 serves as a membranous, cytosolic marker and a mitochondrial marker, respectively. Cyt. and Mit. represent the cytosolic fraction without membranous organelles and the mitochondrial fraction, respectively. B. Proteinase K digestion assay for mitochondria fractions of H1299 cells. The intact mitochondria of H1299 was extracted and then incubated with the indicated concentration of PK at 25°C for 30 min. The fraction with PK and 0.1% of Triton X-100 serves as the negative control. After incubation, the mixture was subjected to immunoblotting. Tom20 and Tim23 serve as the markers for the outer and inner membrane of the mitochondria, respectively. The upper right panel showed the relative protein level digested by PK. C. H1299 cells were starved for 24 h, treated with40 ng/ml for 10 min., and then processed for immunoelectron microscopy analysis. White arrows indicate positive EGFR staining in the mitochondria. Scale bar: 0.2 μm. D. After serum starvation for 24 h, A549 and H1299 cells were treated with 40 ng/ml of EGF at 37°C for 30 min, and then subjected to the mitochondrial fractionation and immunoblotting. E. Starved H1299 cells were pretreated with 0.5 mM of chloroquine for 3 h at 37°C, and then treated with 40 ng/ml of EGF for 15 min. Then the treated cells were subjected into the mitochondrial fractionation and immunoblotting. F. H1299 cells were seeded on the coverslips and serum starved for 24 h. Cells were treated with 40 ng/ml of EGF and 200 nM of MitoTracker for 10 min. Anti-EGFR antibody was used for the immunofluorescence staining. The panels 1.1 and 2.1 are the magnified images of the square in the left panels. The panel 2.2 shows the signals of the colocalization of MitoTracker and EGFR analyzed by ZEN2009 software. Scale bar: 5 μm. G. H1299 cells were serum starved for 24 h and then treated with 200 ng/ml of EGF-Alexa Fluor 488 and 200 nM of MitoTracker for 10 min. The panel F.1 is the magnified image of the square in the left panels. The panel F.2 shows the signals of the colocalization of MitoTracker and EGF-Alexa Fluor 488 analyzed by ZEN2009 software. Scale bar: 5 μm.

    Journal: Oncotarget

    Article Title: Mitochondrial translocation of EGFR regulates mitochondria dynamics and promotes metastasis in NSCLC

    doi:

    Figure Lengend Snippet: EGFR exists in the mitochondria of NSCLC cells and translocates through endocytosis A. Western blotting of the subcellular fractions of CL1–0 and CL1–5 cells. Vinculin and MTCO1 serves as a membranous, cytosolic marker and a mitochondrial marker, respectively. Cyt. and Mit. represent the cytosolic fraction without membranous organelles and the mitochondrial fraction, respectively. B. Proteinase K digestion assay for mitochondria fractions of H1299 cells. The intact mitochondria of H1299 was extracted and then incubated with the indicated concentration of PK at 25°C for 30 min. The fraction with PK and 0.1% of Triton X-100 serves as the negative control. After incubation, the mixture was subjected to immunoblotting. Tom20 and Tim23 serve as the markers for the outer and inner membrane of the mitochondria, respectively. The upper right panel showed the relative protein level digested by PK. C. H1299 cells were starved for 24 h, treated with40 ng/ml for 10 min., and then processed for immunoelectron microscopy analysis. White arrows indicate positive EGFR staining in the mitochondria. Scale bar: 0.2 μm. D. After serum starvation for 24 h, A549 and H1299 cells were treated with 40 ng/ml of EGF at 37°C for 30 min, and then subjected to the mitochondrial fractionation and immunoblotting. E. Starved H1299 cells were pretreated with 0.5 mM of chloroquine for 3 h at 37°C, and then treated with 40 ng/ml of EGF for 15 min. Then the treated cells were subjected into the mitochondrial fractionation and immunoblotting. F. H1299 cells were seeded on the coverslips and serum starved for 24 h. Cells were treated with 40 ng/ml of EGF and 200 nM of MitoTracker for 10 min. Anti-EGFR antibody was used for the immunofluorescence staining. The panels 1.1 and 2.1 are the magnified images of the square in the left panels. The panel 2.2 shows the signals of the colocalization of MitoTracker and EGFR analyzed by ZEN2009 software. Scale bar: 5 μm. G. H1299 cells were serum starved for 24 h and then treated with 200 ng/ml of EGF-Alexa Fluor 488 and 200 nM of MitoTracker for 10 min. The panel F.1 is the magnified image of the square in the left panels. The panel F.2 shows the signals of the colocalization of MitoTracker and EGF-Alexa Fluor 488 analyzed by ZEN2009 software. Scale bar: 5 μm.

    Article Snippet: The primary antibodies against EGFR and Tom20 were obtained from Santa Cruz Technology (CA, USA).

    Techniques: Western Blot, Marker, Incubation, Concentration Assay, Negative Control, Immuno-Electron Microscopy, Staining, Fractionation, Immunofluorescence, Software

    Expression of c-erbB2 and EGFR. The HNGC-1 cell line was not immunopositive for c-erbB2, as assayed by immunostaining with c-erbB2 antibody (A). About 30% to 40% of the cells from the HNGC-2 cell line showed a strong membranous staining for c-erbB2 (B) and this was quantitatively assayed by flow cytometry wherein, as demonstrated in a representative experiment, a distinct peak of erbB2 + cells is observed with a percent positivity of 35% and an MFI of 38 above a negative cutoff with isotype control of 5% (C). HNGC-2 expressed a 170-kDa EGFR band in Western blotting (D). The expression of c-erbB2 as a 185-kDa protein (E) in the HNGC-2 cell line was comparable to that of the A431 cell line used as a positive control. Equal loading of protein was confirmed by using β-actin loading controls for the two cell lines A431(f) and HNGC-2.

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

    Article Title: A Unique Model System for Tumor Progression in GBM Comprising Two Developed Human Neuro-Epithelial Cell Lines with Differential Transforming Potential and Coexpressing Neuronal and Glial Markers 1

    doi:

    Figure Lengend Snippet: Expression of c-erbB2 and EGFR. The HNGC-1 cell line was not immunopositive for c-erbB2, as assayed by immunostaining with c-erbB2 antibody (A). About 30% to 40% of the cells from the HNGC-2 cell line showed a strong membranous staining for c-erbB2 (B) and this was quantitatively assayed by flow cytometry wherein, as demonstrated in a representative experiment, a distinct peak of erbB2 + cells is observed with a percent positivity of 35% and an MFI of 38 above a negative cutoff with isotype control of 5% (C). HNGC-2 expressed a 170-kDa EGFR band in Western blotting (D). The expression of c-erbB2 as a 185-kDa protein (E) in the HNGC-2 cell line was comparable to that of the A431 cell line used as a positive control. Equal loading of protein was confirmed by using β-actin loading controls for the two cell lines A431(f) and HNGC-2.

    Article Snippet: Immunodetection was performed using 1:100 dilution of c-erbB2 and EGFR antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 3 hours at 4°C.

    Techniques: Expressing, Immunostaining, Staining, Flow Cytometry, Cytometry, Western Blot, Positive Control

    Expression of thymidylate synthase (TS) and related EGFR signaling pathway proteins in tumor xenografts after the administration of different treatments ( n = 6 mice/group). Groups: control; Ico, icotinib; Ico‐Pem, sequential Ico followed by Pem; Ico + Pem, concurrent Ico and Pem; Pem, pemetrexed; Pem‐Ico, sequential Pem followed by Ico. ( a ) The effects of different combinations of Ico and Pem on TS expression and EGFR, AKT, and MAPK phosphorylation in tumor tissues was detected by Western blotting. The relative ( b ) TS ( c ) phospho‐EGFR, ( d ) phospho‐AKT, and ( e ) phospho‐MAPK expression levels. Data are shown as the mean ± standard deviation of triplicate measurements. P

    Journal: Thoracic Cancer

    Article Title: Efficacy of combined icotinib and pemetrexed in EGFR mutant lung adenocarcinoma cell line xenografts

    doi: 10.1111/1759-7714.12818

    Figure Lengend Snippet: Expression of thymidylate synthase (TS) and related EGFR signaling pathway proteins in tumor xenografts after the administration of different treatments ( n = 6 mice/group). Groups: control; Ico, icotinib; Ico‐Pem, sequential Ico followed by Pem; Ico + Pem, concurrent Ico and Pem; Pem, pemetrexed; Pem‐Ico, sequential Pem followed by Ico. ( a ) The effects of different combinations of Ico and Pem on TS expression and EGFR, AKT, and MAPK phosphorylation in tumor tissues was detected by Western blotting. The relative ( b ) TS ( c ) phospho‐EGFR, ( d ) phospho‐AKT, and ( e ) phospho‐MAPK expression levels. Data are shown as the mean ± standard deviation of triplicate measurements. P

    Article Snippet: After blocking with 5% bovine serum albumin at room temperature for one hour, primary antibodies including anti‐TS, anti‐EGFR, anti‐phospho‐EGFR, anti‐AKT, anti‐phospho‐AKT, anti‐MAPK, anti‐phospho‐MAPK, and anti‐β‐actin antibody (Abcam, Cambridge, MA, USA) were added overnight and incubated at 4°C.

    Techniques: Expressing, Mouse Assay, End-sequence Profiling, Western Blot, Standard Deviation

    Both PI3K/Akt and MAPK signaling pathways are essential in lung cancer cells harboring wild-type EGFR ( A ) PI3K inhibition suppressed proliferation of lung cancer cells harboring mutant or wild-type EGFR . A549 and PC-9 cells were incubated in the presence of various concentrations of GSK2126458 or PF04691502. Cell extracts were prepared after 1 h of treatment and immunoblotted with the indicated antibodies (upper panel). Cell growth was measured by MTT assay after 72 h (lower panel). ( B ) MEK inhibition suppressed proliferation of lung cancer cells harboring mutant or wild-type EGFR . A549 and PC-9 cells were incubated in the presence of various concentrations of AZD-8330 or TAK-733. Cell extracts were prepared after 1 h of treatment and immunoblotted with the indicated antibodies (upper panel). Cell growth was measured by MTT assay after 72 h (lower panel).

    Journal: Oncotarget

    Article Title: Hepatocyte growth factor reduces sensitivity to the epidermal growth factor receptor-tyrosine kinase inhibitor, gefitinib, in lung adenocarcinoma cells harboring wild-type EGFR

    doi: 10.18632/oncotarget.7586

    Figure Lengend Snippet: Both PI3K/Akt and MAPK signaling pathways are essential in lung cancer cells harboring wild-type EGFR ( A ) PI3K inhibition suppressed proliferation of lung cancer cells harboring mutant or wild-type EGFR . A549 and PC-9 cells were incubated in the presence of various concentrations of GSK2126458 or PF04691502. Cell extracts were prepared after 1 h of treatment and immunoblotted with the indicated antibodies (upper panel). Cell growth was measured by MTT assay after 72 h (lower panel). ( B ) MEK inhibition suppressed proliferation of lung cancer cells harboring mutant or wild-type EGFR . A549 and PC-9 cells were incubated in the presence of various concentrations of AZD-8330 or TAK-733. Cell extracts were prepared after 1 h of treatment and immunoblotted with the indicated antibodies (upper panel). Cell growth was measured by MTT assay after 72 h (lower panel).

    Article Snippet: The primary antibodies used in this study were anti-Met (25H2), anti–phospho-Met (Y1234/Y1235) (3D7), anti–phospho EGFR (Y1068), anti-ErbB3 (1B2), anti–phospho-ErbB3 (Tyr1289) (21D3), anti-Akt, or phospho-Akt (Ser473) antibodies (1:1,000 dilution, Cell Signaling Technology), anti-human EGFR (1 μg/mL), anti-human/mouse/rat extracellular signal regulated kinase (ERK)-1/ERK2 (0.2 μg/mL), and anti–phospho ERK1/ERK2 (T202/Y204) (0.1 μg/mL) antibodies (R & D Systems).

    Techniques: Inhibition, Mutagenesis, Incubation, MTT Assay

    Effect of GM3 on EGF-induced tyrosine phosphorylation of EGFR. Notes: ( A ) Technique (schematic) for evaluating interaction of GM 3 -magnetosomes with EGFR. ( B ) A431 cell lysate was incubated with PE-magnetosomes or GM 3 -magnetosomes, separated by a magnetic field, boiled in 50 µL sample buffer, subjected to SDS-PAGE, visualized by silver staining (left), or analyzed by Western blotting (right). Lane 1: A431 cell lysate. Lane 2: lysate incubated with PE-magnetosomes. Lane 3: lysate incubated with GM 3 -magnetosomes. Lane 4: protein bound to PE-magnetosomes. Lane 5: protein bound to GM 3 -magnetosomes. ( C ) Inhibitory effect of GM 3 and GM 3 -magnetosomes on EGFR kinase activity in A431 cells. Amount of GM 3 per 1 mg magnetosome =0.104 µg. 10*: concentration of total GM 3 -magnetosome complex added to medium =10 µg/mL. EGF-induced phosphorylation of EGFR was quantified as density of phospho-EGFR divided by that of EGFR, by densitometry using Image J. Experiments were performed in triplicate, and representative Western blot results are shown (left). Data are shown as mean ± SD (right). * P

    Journal: International Journal of Nanomedicine

    Article Title: Ganglioside-magnetosome complex formation enhances uptake of gangliosides by cells

    doi: 10.2147/IJN.S92228

    Figure Lengend Snippet: Effect of GM3 on EGF-induced tyrosine phosphorylation of EGFR. Notes: ( A ) Technique (schematic) for evaluating interaction of GM 3 -magnetosomes with EGFR. ( B ) A431 cell lysate was incubated with PE-magnetosomes or GM 3 -magnetosomes, separated by a magnetic field, boiled in 50 µL sample buffer, subjected to SDS-PAGE, visualized by silver staining (left), or analyzed by Western blotting (right). Lane 1: A431 cell lysate. Lane 2: lysate incubated with PE-magnetosomes. Lane 3: lysate incubated with GM 3 -magnetosomes. Lane 4: protein bound to PE-magnetosomes. Lane 5: protein bound to GM 3 -magnetosomes. ( C ) Inhibitory effect of GM 3 and GM 3 -magnetosomes on EGFR kinase activity in A431 cells. Amount of GM 3 per 1 mg magnetosome =0.104 µg. 10*: concentration of total GM 3 -magnetosome complex added to medium =10 µg/mL. EGF-induced phosphorylation of EGFR was quantified as density of phospho-EGFR divided by that of EGFR, by densitometry using Image J. Experiments were performed in triplicate, and representative Western blot results are shown (left). Data are shown as mean ± SD (right). * P

    Article Snippet: Protein in the supernatant (equal amount for each sample) was subjected to SDS-PAGE (7% gel) and Western blotting with primary anti-phosphorylated EGFR (pEGFR; dilution 1:1,000; Cell Signaling Technology) or anti-EGFR (dilution 1:1,000) antibody and horseradish peroxidase-conjugated anti-rabbit secondary antibody.

    Techniques: Incubation, SDS Page, Silver Staining, Western Blot, Activity Assay, Concentration Assay

    EP2 overexpression increases Src phosphorylation and activity. (A) EP2 overexpression increases Src phosphorylation. HEK 293 cells were transfected with empty vector or pcDNA3.1(−) EP2 and pcDNA3-Src. Cell lysates were subjected to western blot analysis for p-Src (Tyr416). Membranes were stripped and reprobed for Src to confirm equal expression of Src. (B) EP2 overexpression increases endogenous Src activity. HEK 293 cells were transfected with empty vector or pcDNA3.1(−) EP2 and pcDNA3-EGFR. Twenty-four hours after transfection, cells were incubated with or without PP2 for another 24 hours. Cell lysates were subjected to western blot analysis for p-EGFR (Tyr845). Membranes were stripped and reprobed for EGFR to confirm equal expression of EGFR. Additionally, cell lysates were subjected to western blot analysis for p-Src and total Src to examine the effect of PP2 on endogenous Src phosphorylation in HEK 293 cells.

    Journal: Biomolecules & Therapeutics

    Article Title: EP2 Induces p38 Phosphorylation via the Activation of Src in HEK 293 Cells

    doi: 10.4062/biomolther.2015.043

    Figure Lengend Snippet: EP2 overexpression increases Src phosphorylation and activity. (A) EP2 overexpression increases Src phosphorylation. HEK 293 cells were transfected with empty vector or pcDNA3.1(−) EP2 and pcDNA3-Src. Cell lysates were subjected to western blot analysis for p-Src (Tyr416). Membranes were stripped and reprobed for Src to confirm equal expression of Src. (B) EP2 overexpression increases endogenous Src activity. HEK 293 cells were transfected with empty vector or pcDNA3.1(−) EP2 and pcDNA3-EGFR. Twenty-four hours after transfection, cells were incubated with or without PP2 for another 24 hours. Cell lysates were subjected to western blot analysis for p-EGFR (Tyr845). Membranes were stripped and reprobed for EGFR to confirm equal expression of EGFR. Additionally, cell lysates were subjected to western blot analysis for p-Src and total Src to examine the effect of PP2 on endogenous Src phosphorylation in HEK 293 cells.

    Article Snippet: Antibodies that were used are phosphor (p)-p38 (Cell Signaling Technology), p-Hsp27 (Cell Signaling Technology), p-Erk (Cell Signaling Technology), p-JNK (Cell Signaling Technology), p-Src Family (Cell Signaling Technology), Src (Cell Signaling Technology), p-EGFR (Cell Signaling Technology), EGFR (Cell Signaling Technology), anti-FLAG® M2-HRP (Sigma-Aldrich, St Louis, MO, USA), anti-c-Myc-HRP (GenScript, Piscataway, NJ, USA), and anti-HA-HRP (Roche Life Sciences, Indianpolis, USA).

    Techniques: Over Expression, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Incubation

    ADAM8 preferentially localizes to anchoring columns of placental villi. ( A ) Representative immunofluorescence, immunohistochemistry and RNAScope microscopy images of serially sectioned first trimester placental villi (6–11 weeks gestation; n = 5) and decidua (10–12 weeks gestation; n = 4) showing ADAM8 mRNA transcript localization (pink punctate signal) within trophoblast subtypes. Proximal and distal column, as well as interstitial EVT subtypes of trophoblasts are identified by immunostaining of cytokeratin (KRT7) and HLA-G. ‘PCT’ indicates proximal column trophoblast; ‘DCT’ indicates distal column trophoblast; ‘VT’ indicates villous trophoblast; ‘SynT’ indicates syncytiotrophoblast; ‘MC’ indicates placenta mesenchymal core; ‘iEVT’ indicates interstitial extravillous trophoblast; ‘GC’ indicates trophoblast giant cell. The perforated white box indicates enlarged inset image. Black arrowheads denote ADAM8 signal. Bars = 100 μm. ( B ) Fluorescence activated cell-sorting (FACS) plots demonstrate the trophoblast isolation strategy used to purify mesencymal core cells (MC), distal column trophoblasts (DCT) and villous/proximal column trophoblasts (VT). Live cells, depleted of CD31 + (endothelial) and CD45 + (immune) cells using immuno-magnetic beads, were positively gated by 7AAD exclusion. Cells were further segregated by excluding CD45 + immune cells and by cell surface labeling of HLA-G and CD49f. Cell subtype proportions are indicated within each gated population (percent of cells within FACS plot). ( C ) ADAM8 mRNA levels in FACS-purified MCs, VTs and DCTs. Trophoblast subtype purity was assessed by qPCR analysis targeting the pan-trophoblast marker KRT7 , the EVT-marker HLA-G and the mesenchymal lineage marker VIM . GAPDH was used for normalization. Results are presented as mean ± SD in bar graphs from four distinct placental villi specimens ( n = 4); results were analyzed by one-way ANOVA and Dunn’s multiple comparisons test. ( D ) Immunoblot showing protein levels of pro- and active-ADAM8, HLA-G, and EGFR in MC, VT and DCT isolated from placental villi using immuno-magnetic beads. Molecular weights (kDa) are shown to the left and β-actin indicates loading control.

    Journal: Molecular Human Reproduction

    Article Title: ADAM8 localizes to extravillous trophoblasts within the maternal–fetal interface and potentiates trophoblast cell line migration through a β1 integrin-mediated mechanism

    doi: 10.1093/molehr/gay034

    Figure Lengend Snippet: ADAM8 preferentially localizes to anchoring columns of placental villi. ( A ) Representative immunofluorescence, immunohistochemistry and RNAScope microscopy images of serially sectioned first trimester placental villi (6–11 weeks gestation; n = 5) and decidua (10–12 weeks gestation; n = 4) showing ADAM8 mRNA transcript localization (pink punctate signal) within trophoblast subtypes. Proximal and distal column, as well as interstitial EVT subtypes of trophoblasts are identified by immunostaining of cytokeratin (KRT7) and HLA-G. ‘PCT’ indicates proximal column trophoblast; ‘DCT’ indicates distal column trophoblast; ‘VT’ indicates villous trophoblast; ‘SynT’ indicates syncytiotrophoblast; ‘MC’ indicates placenta mesenchymal core; ‘iEVT’ indicates interstitial extravillous trophoblast; ‘GC’ indicates trophoblast giant cell. The perforated white box indicates enlarged inset image. Black arrowheads denote ADAM8 signal. Bars = 100 μm. ( B ) Fluorescence activated cell-sorting (FACS) plots demonstrate the trophoblast isolation strategy used to purify mesencymal core cells (MC), distal column trophoblasts (DCT) and villous/proximal column trophoblasts (VT). Live cells, depleted of CD31 + (endothelial) and CD45 + (immune) cells using immuno-magnetic beads, were positively gated by 7AAD exclusion. Cells were further segregated by excluding CD45 + immune cells and by cell surface labeling of HLA-G and CD49f. Cell subtype proportions are indicated within each gated population (percent of cells within FACS plot). ( C ) ADAM8 mRNA levels in FACS-purified MCs, VTs and DCTs. Trophoblast subtype purity was assessed by qPCR analysis targeting the pan-trophoblast marker KRT7 , the EVT-marker HLA-G and the mesenchymal lineage marker VIM . GAPDH was used for normalization. Results are presented as mean ± SD in bar graphs from four distinct placental villi specimens ( n = 4); results were analyzed by one-way ANOVA and Dunn’s multiple comparisons test. ( D ) Immunoblot showing protein levels of pro- and active-ADAM8, HLA-G, and EGFR in MC, VT and DCT isolated from placental villi using immuno-magnetic beads. Molecular weights (kDa) are shown to the left and β-actin indicates loading control.

    Article Snippet: The membranes were probed using antibodies directed against rabbit anti-ADAM8 (polyclonal, 1:1000, LSBio), rabbit β1-integrin (polyclonal, 1:1000, Cell Signaling), mouse anti-HA (clone HA-7, 1:1000, BioLegend, San Diego, CA, USA), mouse anti-Myc (clone Myc.A7, 1:5000, Sigma Aldrich), mouse anti-HLA-G (clone 4H84, 1:2000, ExBio), rabbit anti-EGFR (clone D38B1, 1:1000, Cell Signaling), mouse anti-GAPDH (clone 6C5, 1:10 000, HyTest, Turku, Finland).

    Techniques: Immunofluorescence, Immunohistochemistry, Microscopy, Immunostaining, Fluorescence, FACS, Isolation, Magnetic Beads, Labeling, Purification, Real-time Polymerase Chain Reaction, Marker

    ADAM8 increases during extravillous trophoblast (EVT) differentiation and promotes cell migration. ( A ) Representative immunofluorescence microscopy image of primary trophoblasts 24 h post-seeding onto fibronectin-coated plate. Cells were immunostained with antibodies directed against keratin-7 (green), vimentin (red) and DAPI (blue). Bar = 100 μm. ( B ) Immunoblot showing protein levels of pro- and active-ADAM8, EGFR and HLA-G in whole cell lysates derived from primary trophoblasts cultured over 72 h on fibronectin-coated plates. Molecular weights (kDa) are shown to the left and β-actin indicates loading control. ( C ) Graph depicts pair-wise comparisons of pro- and active-ADAM8 protein fold-change in primary trophoblasts at 72 h of culture compared to levels at 24 h ( n = 3). Shown is the P value (paired t test, two-tailed). ( D ) Representative immunoblot showing protein levels of pro- and active-ADAM8 and HLA-G in primary trophoblasts transfected with control non-silencing siRNA (NS) or siRNA targeting ADAM8 (A8i-1, A8i-2) following 72 h of culture ( n = 3). Molecular weights (kDa) are shown to the left and GAPDH indicates loading control. ( E ) Representative Transwell migration images of primary trophoblasts transfected with control or ADAM8-silencing siRNA stained with DAPI (white) following 36 h of culture ( n = 3 experiments). Graph to the right shows quantification of trophoblast migration as a proportion normalized to NS control. * P ≤ 0.05, ** P ≤ 0.01; one-way ANOVA (Dunn’s multiple comparisons test).

    Journal: Molecular Human Reproduction

    Article Title: ADAM8 localizes to extravillous trophoblasts within the maternal–fetal interface and potentiates trophoblast cell line migration through a β1 integrin-mediated mechanism

    doi: 10.1093/molehr/gay034

    Figure Lengend Snippet: ADAM8 increases during extravillous trophoblast (EVT) differentiation and promotes cell migration. ( A ) Representative immunofluorescence microscopy image of primary trophoblasts 24 h post-seeding onto fibronectin-coated plate. Cells were immunostained with antibodies directed against keratin-7 (green), vimentin (red) and DAPI (blue). Bar = 100 μm. ( B ) Immunoblot showing protein levels of pro- and active-ADAM8, EGFR and HLA-G in whole cell lysates derived from primary trophoblasts cultured over 72 h on fibronectin-coated plates. Molecular weights (kDa) are shown to the left and β-actin indicates loading control. ( C ) Graph depicts pair-wise comparisons of pro- and active-ADAM8 protein fold-change in primary trophoblasts at 72 h of culture compared to levels at 24 h ( n = 3). Shown is the P value (paired t test, two-tailed). ( D ) Representative immunoblot showing protein levels of pro- and active-ADAM8 and HLA-G in primary trophoblasts transfected with control non-silencing siRNA (NS) or siRNA targeting ADAM8 (A8i-1, A8i-2) following 72 h of culture ( n = 3). Molecular weights (kDa) are shown to the left and GAPDH indicates loading control. ( E ) Representative Transwell migration images of primary trophoblasts transfected with control or ADAM8-silencing siRNA stained with DAPI (white) following 36 h of culture ( n = 3 experiments). Graph to the right shows quantification of trophoblast migration as a proportion normalized to NS control. * P ≤ 0.05, ** P ≤ 0.01; one-way ANOVA (Dunn’s multiple comparisons test).

    Article Snippet: The membranes were probed using antibodies directed against rabbit anti-ADAM8 (polyclonal, 1:1000, LSBio), rabbit β1-integrin (polyclonal, 1:1000, Cell Signaling), mouse anti-HA (clone HA-7, 1:1000, BioLegend, San Diego, CA, USA), mouse anti-Myc (clone Myc.A7, 1:5000, Sigma Aldrich), mouse anti-HLA-G (clone 4H84, 1:2000, ExBio), rabbit anti-EGFR (clone D38B1, 1:1000, Cell Signaling), mouse anti-GAPDH (clone 6C5, 1:10 000, HyTest, Turku, Finland).

    Techniques: Migration, Immunofluorescence, Microscopy, Derivative Assay, Cell Culture, Two Tailed Test, Transfection, Staining

    TGF-α-mediated internalization of EGFR and CD81. (A) Huh-7.5 cells were treated with 5 nM TGF-α in the presence or absence of 5 μM erlotinib for 15 min at 37°C and fixed with paraformaldehyde, and immunofluorescence staining

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.00750-12

    Figure Lengend Snippet: TGF-α-mediated internalization of EGFR and CD81. (A) Huh-7.5 cells were treated with 5 nM TGF-α in the presence or absence of 5 μM erlotinib for 15 min at 37°C and fixed with paraformaldehyde, and immunofluorescence staining

    Article Snippet: For time-of-addition experiments with EGFR ligands, antibodies, or HDL, Huh-7.5 cells were treated with either 12.5 nM TGF-α (US Biologicals, Swampscott, MA), 16 nM EGF (Millipore), 20 μg of anti-EGFR antibodies (cetuximab or D1.5)/ml, or 10 μg of HDL (EMD Chemicals, Inc., Gibbstown, NJ)/ml for various times at 37°C (0, 15, 30, 60, 90, 120, and 240 min).

    Techniques: Immunofluorescence, Staining

    (A to C) Expression and colocalization of EGFR with SR-BI (A), CLDN1 (B), and OCLN (C) in untreated and TGF-α-treated Huh-7.5 cells. Huh-7.5 cells were treated with 5 nM TGF-α for 15 min at 37°C and fixed with paraformaldehyde,

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.00750-12

    Figure Lengend Snippet: (A to C) Expression and colocalization of EGFR with SR-BI (A), CLDN1 (B), and OCLN (C) in untreated and TGF-α-treated Huh-7.5 cells. Huh-7.5 cells were treated with 5 nM TGF-α for 15 min at 37°C and fixed with paraformaldehyde,

    Article Snippet: For time-of-addition experiments with EGFR ligands, antibodies, or HDL, Huh-7.5 cells were treated with either 12.5 nM TGF-α (US Biologicals, Swampscott, MA), 16 nM EGF (Millipore), 20 μg of anti-EGFR antibodies (cetuximab or D1.5)/ml, or 10 μg of HDL (EMD Chemicals, Inc., Gibbstown, NJ)/ml for various times at 37°C (0, 15, 30, 60, 90, 120, and 240 min).

    Techniques: Expressing

    Src Family Kinases mediate nEGFR translocation in TNBC (A) Constitutively active Src (caSrc) enhances nEGFR translocation in TNBC cell lines . Cells were transfected with caSrc or an empty vector control for 48 hr prior to stimulation with EGF (5 nM, 45 min) to induce nEGFR translocation. Non-nuclear and nuclear proteins were harvested. nEGFR expression was quantitated using ImageJ software (B) A negative regulator of Src, src-like adaptor protein (SLAP), blocks nEGFR translocation in TNBC cell lines. Cells were transfected with SLAP-FLAG or an empty vector control for 48 hr prior to harvesting non-nuclear and nuclear proteins. nEGFR expression was analyzed. (C) SLAP can interact with EGFR and decrease EGFR activation at Tyrosine 1101. Cells were transfected with SLAP-FLAG or an empty vector control for 48 hr prior to harvesting whole cell lysate. 250 ug of cell lysate was immunoprecipitated with an anti-SLAP antibody. The same lysate was subjected to immunoblot analysis for activation of EGFR at Tyrosine 1101. pEGFR-Y1101 activity was quantitated using ImageJ software. Inset 1: EGFR mutated at Tyrosine 1101 is deficient in nuclear localization. Vector, EGFR-WT, and EGFR-Y1101F were transfected into CHOK1 cells for 48 hr prior to stimulation with EGF (5 nM, 45 min). Non-nuclear and nuclear proteins were harvested, and nEGFR expression was analyzed.

    Journal: Molecular cancer therapeutics

    Article Title: Nuclear Epidermal Growth Factor Receptor is a Functional Molecular Target in Triple-Negative Breast Cancer

    doi: 10.1158/1535-7163.MCT-13-1021

    Figure Lengend Snippet: Src Family Kinases mediate nEGFR translocation in TNBC (A) Constitutively active Src (caSrc) enhances nEGFR translocation in TNBC cell lines . Cells were transfected with caSrc or an empty vector control for 48 hr prior to stimulation with EGF (5 nM, 45 min) to induce nEGFR translocation. Non-nuclear and nuclear proteins were harvested. nEGFR expression was quantitated using ImageJ software (B) A negative regulator of Src, src-like adaptor protein (SLAP), blocks nEGFR translocation in TNBC cell lines. Cells were transfected with SLAP-FLAG or an empty vector control for 48 hr prior to harvesting non-nuclear and nuclear proteins. nEGFR expression was analyzed. (C) SLAP can interact with EGFR and decrease EGFR activation at Tyrosine 1101. Cells were transfected with SLAP-FLAG or an empty vector control for 48 hr prior to harvesting whole cell lysate. 250 ug of cell lysate was immunoprecipitated with an anti-SLAP antibody. The same lysate was subjected to immunoblot analysis for activation of EGFR at Tyrosine 1101. pEGFR-Y1101 activity was quantitated using ImageJ software. Inset 1: EGFR mutated at Tyrosine 1101 is deficient in nuclear localization. Vector, EGFR-WT, and EGFR-Y1101F were transfected into CHOK1 cells for 48 hr prior to stimulation with EGF (5 nM, 45 min). Non-nuclear and nuclear proteins were harvested, and nEGFR expression was analyzed.

    Article Snippet: SFK (CS2123), pSFK-Y419 (CS2101), pEGFR-Y1045 (CS2237), pEGFR-Y1068 (CS3777), pHER2-Y1221/1222 (CS2243), c-Cbl (CS2747), GAPDH (CS2118), calnexin (CS2679), and anti-Flag (CS8146) purchased from Cell Signaling Technology (Beverly, MA, USA). pEGFR-Y1101 (ab76195) and EGFR (ab52894) purchased from Abcam (Cambridge, MA, USA). α-Tubulin purchased from Calbiochem (San Diego, CA, USA).

    Techniques: Translocation Assay, Transfection, Plasmid Preparation, Expressing, Software, Activation Assay, Immunoprecipitation, Activity Assay

    U87 and U251 cell lines were transfected with si-LRIG2 or si-NC, then treated with 100 ng/ml EGF or DDH 2 O for 24 h. (A) The protein expression level of EGFR, p-EGFR and VEGF-A were assessed by western blot assay. (B) The densitometry data of western blotting. **P

    Journal: Oncology Letters

    Article Title: Downregulation of LRIG2 expression inhibits angiogenesis of glioma via EGFR/VEGF-A pathway

    doi: 10.3892/ol.2017.6671

    Figure Lengend Snippet: U87 and U251 cell lines were transfected with si-LRIG2 or si-NC, then treated with 100 ng/ml EGF or DDH 2 O for 24 h. (A) The protein expression level of EGFR, p-EGFR and VEGF-A were assessed by western blot assay. (B) The densitometry data of western blotting. **P

    Article Snippet: Anti-LRIG2 antibody (ab157492), anti-EGFR antibody (ab52894), anti-VEGF-A antibody (ab46154), anti-CD31 antibody (ab28364), anti-p-EGFR antibody (ab40815) and biotinylated anti-rabbit immunoglobulin G were obtained from Abcam (Cambridge, MA, USA).

    Techniques: Transfection, Expressing, Western Blot

    Downregulation of LRIG2 decreased the expression of EGFR and VEGF-A. (A) The protein expression level of EGFR, p-EGFR and VEGF-A were assessed by western blot assay. (B) The densitometry data of western blotting. **P

    Journal: Oncology Letters

    Article Title: Downregulation of LRIG2 expression inhibits angiogenesis of glioma via EGFR/VEGF-A pathway

    doi: 10.3892/ol.2017.6671

    Figure Lengend Snippet: Downregulation of LRIG2 decreased the expression of EGFR and VEGF-A. (A) The protein expression level of EGFR, p-EGFR and VEGF-A were assessed by western blot assay. (B) The densitometry data of western blotting. **P

    Article Snippet: Anti-LRIG2 antibody (ab157492), anti-EGFR antibody (ab52894), anti-VEGF-A antibody (ab46154), anti-CD31 antibody (ab28364), anti-p-EGFR antibody (ab40815) and biotinylated anti-rabbit immunoglobulin G were obtained from Abcam (Cambridge, MA, USA).

    Techniques: Expressing, Western Blot

    We used immunochemistry assay to analyze the protein expression (evaluated by immunoreactive score) of LRIG2, EGFR, VEGF-A and CD31 in glioma tissues and normal brain tissues. The vessels of tissues were expressed by immunohistochemical staining for CD31 highlight for calculation of MVD. The expression level of LRIG2, EGFR, VEGF-A and CD31 was highly expressed in glioma tissues (A), but was not expressed or lowly expressed in normal tissues (B). The expression of LRIG2, EGFR, VEGF-A was directly correlated with MVD. (C) Results are representative of three different experiments. Original magnification, ×400. LRIG2, leucine-rich repeats and immunoglobulin-like domains 2; EGFR, epidermal growth factor receptor; VEGF-A, vascular endothelial growth factor A; CD31, cluster of differentiation 31; MVD, microvessel density.

    Journal: Oncology Letters

    Article Title: Downregulation of LRIG2 expression inhibits angiogenesis of glioma via EGFR/VEGF-A pathway

    doi: 10.3892/ol.2017.6671

    Figure Lengend Snippet: We used immunochemistry assay to analyze the protein expression (evaluated by immunoreactive score) of LRIG2, EGFR, VEGF-A and CD31 in glioma tissues and normal brain tissues. The vessels of tissues were expressed by immunohistochemical staining for CD31 highlight for calculation of MVD. The expression level of LRIG2, EGFR, VEGF-A and CD31 was highly expressed in glioma tissues (A), but was not expressed or lowly expressed in normal tissues (B). The expression of LRIG2, EGFR, VEGF-A was directly correlated with MVD. (C) Results are representative of three different experiments. Original magnification, ×400. LRIG2, leucine-rich repeats and immunoglobulin-like domains 2; EGFR, epidermal growth factor receptor; VEGF-A, vascular endothelial growth factor A; CD31, cluster of differentiation 31; MVD, microvessel density.

    Article Snippet: Anti-LRIG2 antibody (ab157492), anti-EGFR antibody (ab52894), anti-VEGF-A antibody (ab46154), anti-CD31 antibody (ab28364), anti-p-EGFR antibody (ab40815) and biotinylated anti-rabbit immunoglobulin G were obtained from Abcam (Cambridge, MA, USA).

    Techniques: Expressing, Immunohistochemistry, Staining

    Immunohistochemical analysis of SW620CE2 cells growing in the cecum of nude mice. Double immunofluorescence staining was used for CD31/PECAM-1 and EGFR, pEGFR, VEGFR2, or pVEGFR2. (A) The SW620CE2 cells expressed TGF-α and VEGF but not EGFR or VEGFR. The KM12C cells expressed TGF-α and EGFR (green), and the SKOV3ip1 cells (growing in the peritoneal cavity) expressed VEGF and VEGFR2 (green). In all tumors, the endothelial cells expressed EGFR and VEGFR2 (yellow). (B) Colocalization for VEGFR1 and CD31 (endothelial cells) and F4/80 (macrophages) in SW620CE2 cecal tumors. (C) Expression of HER2 (green) in SKOV3ipl but not SW620CE2 tumors.

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

    Article Title: Inhibition of Epidermal Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor Phosphorylation on Tumor-Associated Endothelial Cells Leads to Treatment of Orthotopic Human Colon Cancer in Nude Mice 1

    doi:

    Figure Lengend Snippet: Immunohistochemical analysis of SW620CE2 cells growing in the cecum of nude mice. Double immunofluorescence staining was used for CD31/PECAM-1 and EGFR, pEGFR, VEGFR2, or pVEGFR2. (A) The SW620CE2 cells expressed TGF-α and VEGF but not EGFR or VEGFR. The KM12C cells expressed TGF-α and EGFR (green), and the SKOV3ip1 cells (growing in the peritoneal cavity) expressed VEGF and VEGFR2 (green). In all tumors, the endothelial cells expressed EGFR and VEGFR2 (yellow). (B) Colocalization for VEGFR1 and CD31 (endothelial cells) and F4/80 (macrophages) in SW620CE2 cecal tumors. (C) Expression of HER2 (green) in SKOV3ipl but not SW620CE2 tumors.

    Article Snippet: The following primary antibodies were purchased: rabbit anti-EGFR (SC03; Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-phosphorylated EGFR (pEGFR) (Tyr1173; Biosource, Camarillo, CA); rabbit anti-VEGFR2 (Flk-1, C1158; Santa Cruz Biotechnology); rabbit anti-phosphorylated VEGFR2 (pVEGFR2) (Flk-1, PC460; Oncogene, Boston, MA); rabbit anti-VEGFR1 (C17; Santa Cruz Biotechnology); rabbit anti-mouse F4/80 (MCAP497; Serotec, Raleigh, NC) and rat anti-mouse CD31 (BD PharMingen, San Diego, CA) for frozen samples; mouse anti-human TGF-α (GF10; Oncogene); mouse anti-human VEGF (23071D; BD Phar-Mingen) and Ki-67 (MIB-1; DakoCytomation, Carpinteria, CA) for paraffin-embedded samples; and rabbit anti-EGFR (SC03; Santa Cruz Biotechnology), rabbit anti-pEGFR (Tyr1068; Cell Signaling Technology, Danvers, MA), and rabbit anti-VEGFR2 (Flk-1, C1158; Santa Cruz Biotechnology) for Western blot analysis.

    Techniques: Immunohistochemistry, Mouse Assay, Double Immunofluorescence Staining, Expressing

    Double immunofluorescence staining for expression of EGFR, pEGFR, VEGFR, or pVEGFR in tumor-associated endothelial cells. (A) Tissue sections were stained with anti -CD31/PECAM1 antibody (red) and with anti-EGFR, pEGFR, VEGFR, or pVEGFR (green) as described in the Materials and Methods section. Colocalization of CD31 and EGFR, pEGFR, VEGFR, or pVEGFR appears as yellow fluorescence. Expression of EGFR and VEGFR by tumor-associated endothelial cells was found in tumors from all treatment groups. Phosphorylation of EGFR and VEGFR on endothelial cells was inhibited by treatment with AEE788 or AEE788 plus CPT-11. Note that treatment with AEE788 alone decreased the number and diameter of blood vessels and that this effect was even more pronounced with combined AEE788 and CPT-11.

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

    Article Title: Inhibition of Epidermal Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor Phosphorylation on Tumor-Associated Endothelial Cells Leads to Treatment of Orthotopic Human Colon Cancer in Nude Mice 1

    doi:

    Figure Lengend Snippet: Double immunofluorescence staining for expression of EGFR, pEGFR, VEGFR, or pVEGFR in tumor-associated endothelial cells. (A) Tissue sections were stained with anti -CD31/PECAM1 antibody (red) and with anti-EGFR, pEGFR, VEGFR, or pVEGFR (green) as described in the Materials and Methods section. Colocalization of CD31 and EGFR, pEGFR, VEGFR, or pVEGFR appears as yellow fluorescence. Expression of EGFR and VEGFR by tumor-associated endothelial cells was found in tumors from all treatment groups. Phosphorylation of EGFR and VEGFR on endothelial cells was inhibited by treatment with AEE788 or AEE788 plus CPT-11. Note that treatment with AEE788 alone decreased the number and diameter of blood vessels and that this effect was even more pronounced with combined AEE788 and CPT-11.

    Article Snippet: The following primary antibodies were purchased: rabbit anti-EGFR (SC03; Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-phosphorylated EGFR (pEGFR) (Tyr1173; Biosource, Camarillo, CA); rabbit anti-VEGFR2 (Flk-1, C1158; Santa Cruz Biotechnology); rabbit anti-phosphorylated VEGFR2 (pVEGFR2) (Flk-1, PC460; Oncogene, Boston, MA); rabbit anti-VEGFR1 (C17; Santa Cruz Biotechnology); rabbit anti-mouse F4/80 (MCAP497; Serotec, Raleigh, NC) and rat anti-mouse CD31 (BD PharMingen, San Diego, CA) for frozen samples; mouse anti-human TGF-α (GF10; Oncogene); mouse anti-human VEGF (23071D; BD Phar-Mingen) and Ki-67 (MIB-1; DakoCytomation, Carpinteria, CA) for paraffin-embedded samples; and rabbit anti-EGFR (SC03; Santa Cruz Biotechnology), rabbit anti-pEGFR (Tyr1068; Cell Signaling Technology, Danvers, MA), and rabbit anti-VEGFR2 (Flk-1, C1158; Santa Cruz Biotechnology) for Western blot analysis.

    Techniques: Double Immunofluorescence Staining, Expressing, Staining, Fluorescence, Cycling Probe Technology

    Kinase-mediated ligand-free dimers adopt two ECM dimer architectures. a FLImP distribution (grey) of DIII–DIII separations between CF640R-Affibody molecules bound to wtEGFR on CHO cells treated with 1 μM erlotinib, compiled from 29 FLImP measurements (CI ≤ 6 nm), decomposed into a sum of four components. The concentration of CF640R-Affibody was 4 nM. b Number of measurements consistent with the mean distances resolved in the FLImP distribution of wtEGFR (Fig. 2b ) (associated FLImP distributions in Supplementary Fig. 13 ). Errors were assesed with bootstrap-resampling 12 . c wtEGFR and IIIV/KKRE-EGFR phosphorylation in Y1173 on CHO cells treated or untreated with 10 mM MβCD. Box plots show inclusive median as a line, 25th and 75th quartile as edges, calculated on n = 3 repeats (representative western blots in Supplementary Fig. 14 ). d As a treated with 10 mM MβCD, from 20 FLImP measurements (CI ≤ 7 nm). e Pairwise particle colocalisation fraction from two-colour SPT on live cells at 37 °C. f Duration of pairwise interactions ( τ ON ) in e . Horizontal spreads separate data points (~5000) within each condition. g Crystal structure of tethered wtEGFR (PDB ID 1NQL 5 ) highlighting the location of I545K, I556K, I562R, and V592E (yellow). Colours: DI (green), DII (red), DIII (blue), DIV (grey), EGF (orange). h Head-to-head dimer highlighting the residues mutated in the IIIV/KKRE mutant (yellow). i As a but for the IIIV/KKRE-EGFR mutant from 22 FLImP measurements with CI ≤ 7.5 nm. j FRET-DOCA from DI (blue) and DIII (red) to the membrane for wtEGFR + erlotinib, and L680N-EGFR, derived from measurements in Supplementary Fig. 10 . FRET probes as in Fig. 4e . k Ratio between CF640R-9G8-NB and Alexa 488-EgB4-NB binding after chemical fixation. wtEGFR, blue; wtEGFR + erlotinib, green; L680N-EGFR, red. Line, median; box edges, 25th and 75th quartile, crosses 5th and 95th quartile, calculated over 30 repeats. Example images and analysis are in Supplementary Fig. 15 . l As a but for L680N-EGFR-expressing cells, from 20 FLImP measurements (CI ≤ 6 nm). Lower resolution (8 nm) versions of a , d , and l with ~2-fold more FLImP measurements show the profile of the distributions is conserved (Supplementary Fig. 16 )

    Journal: Nature Communications

    Article Title: The architecture of EGFR’s basal complexes reveals autoinhibition mechanisms in dimers and oligomers

    doi: 10.1038/s41467-018-06632-0

    Figure Lengend Snippet: Kinase-mediated ligand-free dimers adopt two ECM dimer architectures. a FLImP distribution (grey) of DIII–DIII separations between CF640R-Affibody molecules bound to wtEGFR on CHO cells treated with 1 μM erlotinib, compiled from 29 FLImP measurements (CI ≤ 6 nm), decomposed into a sum of four components. The concentration of CF640R-Affibody was 4 nM. b Number of measurements consistent with the mean distances resolved in the FLImP distribution of wtEGFR (Fig. 2b ) (associated FLImP distributions in Supplementary Fig. 13 ). Errors were assesed with bootstrap-resampling 12 . c wtEGFR and IIIV/KKRE-EGFR phosphorylation in Y1173 on CHO cells treated or untreated with 10 mM MβCD. Box plots show inclusive median as a line, 25th and 75th quartile as edges, calculated on n = 3 repeats (representative western blots in Supplementary Fig. 14 ). d As a treated with 10 mM MβCD, from 20 FLImP measurements (CI ≤ 7 nm). e Pairwise particle colocalisation fraction from two-colour SPT on live cells at 37 °C. f Duration of pairwise interactions ( τ ON ) in e . Horizontal spreads separate data points (~5000) within each condition. g Crystal structure of tethered wtEGFR (PDB ID 1NQL 5 ) highlighting the location of I545K, I556K, I562R, and V592E (yellow). Colours: DI (green), DII (red), DIII (blue), DIV (grey), EGF (orange). h Head-to-head dimer highlighting the residues mutated in the IIIV/KKRE mutant (yellow). i As a but for the IIIV/KKRE-EGFR mutant from 22 FLImP measurements with CI ≤ 7.5 nm. j FRET-DOCA from DI (blue) and DIII (red) to the membrane for wtEGFR + erlotinib, and L680N-EGFR, derived from measurements in Supplementary Fig. 10 . FRET probes as in Fig. 4e . k Ratio between CF640R-9G8-NB and Alexa 488-EgB4-NB binding after chemical fixation. wtEGFR, blue; wtEGFR + erlotinib, green; L680N-EGFR, red. Line, median; box edges, 25th and 75th quartile, crosses 5th and 95th quartile, calculated over 30 repeats. Example images and analysis are in Supplementary Fig. 15 . l As a but for L680N-EGFR-expressing cells, from 20 FLImP measurements (CI ≤ 6 nm). Lower resolution (8 nm) versions of a , d , and l with ~2-fold more FLImP measurements show the profile of the distributions is conserved (Supplementary Fig. 16 )

    Article Snippet: We conjugated CF640R fluorophore to the single cysteine of an anti-EGFR Affibody which binds DIII of EGFR’s ECM without activating the receptor and accumulated a FLImP histogram of DIII–DIII separations from ligand-free wtEGFR complexes.

    Techniques: Concentration Assay, Western Blot, Single-particle Tracking, Mutagenesis, Derivative Assay, Binding Assay, Expressing

    Equilibrium between the aTKD and sTKD dimers. a Cartoon of the two dimer configurations of the kinase domain. Starting from the aTKD dimer, a counter clockwise rotation of the activator (blue) along its vertical axis followed by a clockwise rotation along an axis perpendicular to the plane will allow the sTKD to form. b The FLImP distribution (grey) and the distributions compiled from the FLImP measurements whose 69% CIs overlap with DIII–DIII separations = 5 nm (green), 13 nm (yellow), or > 20 nm (blue) collected from cells expressing K721A-EGFR compiled from 29 FLImP measurements with CI ≤ 7 nm. The inset shows the fraction of separations consistent with each distance. Errors were assesed with bootstrap-resampling 12 . c Fraction of tracks where two different-colour particles labelled with Alexa 488-Affibody and CF640-Affibody spent ≥5 frames (250 ms) together within

    Journal: Nature Communications

    Article Title: The architecture of EGFR’s basal complexes reveals autoinhibition mechanisms in dimers and oligomers

    doi: 10.1038/s41467-018-06632-0

    Figure Lengend Snippet: Equilibrium between the aTKD and sTKD dimers. a Cartoon of the two dimer configurations of the kinase domain. Starting from the aTKD dimer, a counter clockwise rotation of the activator (blue) along its vertical axis followed by a clockwise rotation along an axis perpendicular to the plane will allow the sTKD to form. b The FLImP distribution (grey) and the distributions compiled from the FLImP measurements whose 69% CIs overlap with DIII–DIII separations = 5 nm (green), 13 nm (yellow), or > 20 nm (blue) collected from cells expressing K721A-EGFR compiled from 29 FLImP measurements with CI ≤ 7 nm. The inset shows the fraction of separations consistent with each distance. Errors were assesed with bootstrap-resampling 12 . c Fraction of tracks where two different-colour particles labelled with Alexa 488-Affibody and CF640-Affibody spent ≥5 frames (250 ms) together within

    Article Snippet: We conjugated CF640R fluorophore to the single cysteine of an anti-EGFR Affibody which binds DIII of EGFR’s ECM without activating the receptor and accumulated a FLImP histogram of DIII–DIII separations from ligand-free wtEGFR complexes.

    Techniques: Expressing, Mass Spectrometry

    FLImP measurement of pairwise DIII-binding Affibody separations. a Cartoon of FLImP histograms. Left: A dimer (receptors, blue; label, red) gives rise to one separation, the empirical posterior of which has a confidence interval (CI) that depends on the combined localisation errors of the two molecules 28 . CI size determines resolution. CIs less than the required resolution are retained according to signal-to-noise without bias 12 , 28 – 30 , 36 , generating a FLImP distribution (grey) that is fitted by the sum of a number of Rician peaks 12 . If oligomers are present and internally probed (middle) and/or the distribution of species is inhomogeneous (right), the histogram contains multiple components 12 . b FLImP distribution (grey) of DIII–DIII separations between CF640R-Affibody molecules bound to wtEGFR on CHO cells, compiled from 68 FLImP measurements (CI ≤ 7 nm), decomposed into a sum of five components (coloured traces). The inset shows positions and error estimates. Additional statistics in Supplementary Fig. 1 . The 4 nM concentration of CF640R-Affibody used labels ~20% of receptors (Supplementary Fig. 2 ) ensuring single particle detection 29 . FLImP is stochastic, thus independent of the CF640R-Affibody/receptor ratio if sufficient data are collected, and uses fixed cells to avoid relative receptor movements during measurements. However, systematic studies failed to find significant artifacts 12 , 36 . c Molecular-normalised fraction of receptors in oligomer species on wtEGFR-expressing CHO cells treated with 100 nM Alexa 488-Affibody, determined by pbICS 38 . Results are the mean of seven replicates. Error bars show the SD. For more details see Supplementary Fig. 3 . d Cartoon showing expected FLImP distributions for a cyclic tetramer labelled with a 1:1 probe binder (like the Affibody) (left), a polymer chain labelled with a 1:1 probe binder (middle), and a polymer showing a 2N:2 labelling stoichiometry (like EGF) 12 ( N = receptor number) (right). e FLImP distribution (grey) and peak decomposition of DIII–DIII separations reported by CF640R-Affibody molecules bound to I942E-EGFR on the surface of CHO cells, compiled from 36 FLImP measurements (CI ≤ 7 nm), decomposed into a sum of four peak components (coloured traces). Positions and error estimates are shown in the inset. f As e using 4 nM CF640R-EGF as a probe, compiled from 31 FLImP measurements (CI ≤ 7 nm)

    Journal: Nature Communications

    Article Title: The architecture of EGFR’s basal complexes reveals autoinhibition mechanisms in dimers and oligomers

    doi: 10.1038/s41467-018-06632-0

    Figure Lengend Snippet: FLImP measurement of pairwise DIII-binding Affibody separations. a Cartoon of FLImP histograms. Left: A dimer (receptors, blue; label, red) gives rise to one separation, the empirical posterior of which has a confidence interval (CI) that depends on the combined localisation errors of the two molecules 28 . CI size determines resolution. CIs less than the required resolution are retained according to signal-to-noise without bias 12 , 28 – 30 , 36 , generating a FLImP distribution (grey) that is fitted by the sum of a number of Rician peaks 12 . If oligomers are present and internally probed (middle) and/or the distribution of species is inhomogeneous (right), the histogram contains multiple components 12 . b FLImP distribution (grey) of DIII–DIII separations between CF640R-Affibody molecules bound to wtEGFR on CHO cells, compiled from 68 FLImP measurements (CI ≤ 7 nm), decomposed into a sum of five components (coloured traces). The inset shows positions and error estimates. Additional statistics in Supplementary Fig. 1 . The 4 nM concentration of CF640R-Affibody used labels ~20% of receptors (Supplementary Fig. 2 ) ensuring single particle detection 29 . FLImP is stochastic, thus independent of the CF640R-Affibody/receptor ratio if sufficient data are collected, and uses fixed cells to avoid relative receptor movements during measurements. However, systematic studies failed to find significant artifacts 12 , 36 . c Molecular-normalised fraction of receptors in oligomer species on wtEGFR-expressing CHO cells treated with 100 nM Alexa 488-Affibody, determined by pbICS 38 . Results are the mean of seven replicates. Error bars show the SD. For more details see Supplementary Fig. 3 . d Cartoon showing expected FLImP distributions for a cyclic tetramer labelled with a 1:1 probe binder (like the Affibody) (left), a polymer chain labelled with a 1:1 probe binder (middle), and a polymer showing a 2N:2 labelling stoichiometry (like EGF) 12 ( N = receptor number) (right). e FLImP distribution (grey) and peak decomposition of DIII–DIII separations reported by CF640R-Affibody molecules bound to I942E-EGFR on the surface of CHO cells, compiled from 36 FLImP measurements (CI ≤ 7 nm), decomposed into a sum of four peak components (coloured traces). Positions and error estimates are shown in the inset. f As e using 4 nM CF640R-EGF as a probe, compiled from 31 FLImP measurements (CI ≤ 7 nm)

    Article Snippet: We conjugated CF640R fluorophore to the single cysteine of an anti-EGFR Affibody which binds DIII of EGFR’s ECM without activating the receptor and accumulated a FLImP histogram of DIII–DIII separations from ligand-free wtEGFR complexes.

    Techniques: Binding Assay, Concentration Assay, Expressing

    The architecture of ligand-free head-to-head polymers. a Polymer chain formed by repeating the head-to-head interface based on separations in Supplementary Fig. 5a, b and Supplementary Tables 1 and 2 (DII and DIV excluded for simplicity). The intensity is graded according to pbICS results in b , which show the data in Fig. 2c re-normalised to reveal the fractions of oligomer species on wtEGFR-expressing CHO cells treated with 100 nM Alexa 488-Affibody. Results are the mean of seven replicates. Error bars show SD. c FLImP distribution (grey) of DIII–DIII separations in ΔC-EGFR-expressing CHO cells treated with 4 nM CF640R-Affibody, from 41 FLImP measurements (CI ≤ 6 nm). The inset shows positions and error estimates (additional statistics in Supplementary Fig. 7 ). d As c but from ΔC-EGFR-expressing cells treated with 8 nM CF640R-EgB4-NB (DI–DI separations), from 32 FLImP measurements (CI ≤ 8 nm). Differences with the DIII–DIII distribution are significant (Supplementary Fig. 8 ). e Left and centre: Cartoons showing a side view of DI and DIII separations from the membrane in head-to-head complexes in the presence and absence of bound 9G8-NB based on Supplementary Fig. 5c–f and Supplementary Table 3 . Right: FRET-derived separations from the membrane-DiI to DI (Alexa 488-EgB4-NB, blue) or DIII (Alexa 488-Affibody, red). The bar chart was derived from the measurements in Supplementary Fig. 10 . (As predicted by the model, EGFR also forms oligomers on cells treated with 200 nM 9G8-NB (Supplementary Fig. 11a, b ). f Two-colour SPT on live cells at 37 °C showing the fraction of tracks where two particles labelled with Alexa 488-Affibody and CF640R-Affibody spent ≥5 frames (250 ms) together within

    Journal: Nature Communications

    Article Title: The architecture of EGFR’s basal complexes reveals autoinhibition mechanisms in dimers and oligomers

    doi: 10.1038/s41467-018-06632-0

    Figure Lengend Snippet: The architecture of ligand-free head-to-head polymers. a Polymer chain formed by repeating the head-to-head interface based on separations in Supplementary Fig. 5a, b and Supplementary Tables 1 and 2 (DII and DIV excluded for simplicity). The intensity is graded according to pbICS results in b , which show the data in Fig. 2c re-normalised to reveal the fractions of oligomer species on wtEGFR-expressing CHO cells treated with 100 nM Alexa 488-Affibody. Results are the mean of seven replicates. Error bars show SD. c FLImP distribution (grey) of DIII–DIII separations in ΔC-EGFR-expressing CHO cells treated with 4 nM CF640R-Affibody, from 41 FLImP measurements (CI ≤ 6 nm). The inset shows positions and error estimates (additional statistics in Supplementary Fig. 7 ). d As c but from ΔC-EGFR-expressing cells treated with 8 nM CF640R-EgB4-NB (DI–DI separations), from 32 FLImP measurements (CI ≤ 8 nm). Differences with the DIII–DIII distribution are significant (Supplementary Fig. 8 ). e Left and centre: Cartoons showing a side view of DI and DIII separations from the membrane in head-to-head complexes in the presence and absence of bound 9G8-NB based on Supplementary Fig. 5c–f and Supplementary Table 3 . Right: FRET-derived separations from the membrane-DiI to DI (Alexa 488-EgB4-NB, blue) or DIII (Alexa 488-Affibody, red). The bar chart was derived from the measurements in Supplementary Fig. 10 . (As predicted by the model, EGFR also forms oligomers on cells treated with 200 nM 9G8-NB (Supplementary Fig. 11a, b ). f Two-colour SPT on live cells at 37 °C showing the fraction of tracks where two particles labelled with Alexa 488-Affibody and CF640R-Affibody spent ≥5 frames (250 ms) together within

    Article Snippet: We conjugated CF640R fluorophore to the single cysteine of an anti-EGFR Affibody which binds DIII of EGFR’s ECM without activating the receptor and accumulated a FLImP histogram of DIII–DIII separations from ligand-free wtEGFR complexes.

    Techniques: Expressing, Derivative Assay, Single-particle Tracking, Mass Spectrometry

    EGFRvIII–stimulated GBP1 expression is p38 MAPK dependent A. After 24 h of serum starvation, U87-EGFRvIII cells were treated with DMSO (−), 10 μM of the EGFR tyrosine kinase inhibitor AG1478 (AG), or 20μM of the p38 inhibitor SB203580 (SB) for an additional 24 h before Western blot analysis. B. U87-EGFRvIII cells were transfected with the indicated concentration of p38 siRNA (si-p38) or control siRNA (si-Luc) for 24 h and then serum starved for 24 h followed by Western blot analysis. The p38 siRNAs were described previously [ 10 ]. C. U87-EGFRvIII cells were transfected with pGL3-237 and pRL-TK for 24 h and then serum starved for 24 h. The starved cells were pretreated with DMSO or 20 μM SB203580 for an additional 24 h before reporter assay. This result is expressed as the mean of three independent experiments ± SD. *, P

    Journal: Oncotarget

    Article Title: Guanylate binding protein-1 mediates EGFRvIII and promotes glioblastoma growth in vivo but not in vitro

    doi: 10.18632/oncotarget.7109

    Figure Lengend Snippet: EGFRvIII–stimulated GBP1 expression is p38 MAPK dependent A. After 24 h of serum starvation, U87-EGFRvIII cells were treated with DMSO (−), 10 μM of the EGFR tyrosine kinase inhibitor AG1478 (AG), or 20μM of the p38 inhibitor SB203580 (SB) for an additional 24 h before Western blot analysis. B. U87-EGFRvIII cells were transfected with the indicated concentration of p38 siRNA (si-p38) or control siRNA (si-Luc) for 24 h and then serum starved for 24 h followed by Western blot analysis. The p38 siRNAs were described previously [ 10 ]. C. U87-EGFRvIII cells were transfected with pGL3-237 and pRL-TK for 24 h and then serum starved for 24 h. The starved cells were pretreated with DMSO or 20 μM SB203580 for an additional 24 h before reporter assay. This result is expressed as the mean of three independent experiments ± SD. *, P

    Article Snippet: Primary antibodies used were anti-YY1 (c20), anti-p38 (Santa Cruz Biotechnology, Inc.), anti-EGFR (c13; BD), anti–β-actin and –phospho-p38 (Cell Signaling Technology), and anti-GBP1 (MBL International).

    Techniques: Expressing, Western Blot, Transfection, Concentration Assay, Reporter Assay

    Proteasome-dependent degradation of Fhit in 293 cells. ( A ) The 293 cells were transiently transfected with empty vector (lane 1), FHIT (lane 2), or FHIT and SRC (lanes 3 and 4). The sample in lane 4 was from cells pretreated with 15 μM lactacystin. ( B ) The 293 cells were cotransfected with FHIT and EGFR and stimulated with 100 ng/ml of EGF for the indicated time. Cells were pretreated (lanes 7–12) or not (lanes 1–6) with 15 μM lactacystin. Western blot analyses of total lysates were carried out by using Fhit polyclonal antibody (Fhit), a phospho-Fhit polyclonal antibody (P-Fhit), a mAb to phosphotyrosine (PY), a mAb to Src (Src) and a mAb to actin (Actin).

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

    Article Title: FHIT-proteasome degradation caused by mitogenic stimulation of the EGF receptor family in cancer cells

    doi: 10.1073/pnas.0605821103

    Figure Lengend Snippet: Proteasome-dependent degradation of Fhit in 293 cells. ( A ) The 293 cells were transiently transfected with empty vector (lane 1), FHIT (lane 2), or FHIT and SRC (lanes 3 and 4). The sample in lane 4 was from cells pretreated with 15 μM lactacystin. ( B ) The 293 cells were cotransfected with FHIT and EGFR and stimulated with 100 ng/ml of EGF for the indicated time. Cells were pretreated (lanes 7–12) or not (lanes 1–6) with 15 μM lactacystin. Western blot analyses of total lysates were carried out by using Fhit polyclonal antibody (Fhit), a phospho-Fhit polyclonal antibody (P-Fhit), a mAb to phosphotyrosine (PY), a mAb to Src (Src) and a mAb to actin (Actin).

    Article Snippet: Antibodies used were: mouse monoclonal anti-phosphotyrosine (Upstate Biotechnology, Lake Placid, NY); rabbit polyclonal anti-Fhit (Zymed Laboratories, San Francisco, CA); mouse monoclonal anti-Src clone GD11 (Upstate Biotechnology); mouse monoclonal anti-actin clone AC-40 (Sigma); rabbit polyclonal anti-phospho-Fhit-Tyr-114 (Cell Signaling Technology, Danvers, MA); mouse monoclonal anti-Flag M2 (Sigma); rabbit polyclonal anti-p44/42 MAPK (New England Biolabs, Ipswich, MA); mouse monoclonal anti-phospho p44/42 MAPK (Thr-202/Tyr-204) (Cell Signaling Technology); rabbit polyclonal anti-EGFR clone 1005 (Santa Cruz Biotechnology, Santa Cruz, CA); mouse monoclonal anti-EGFR phospho-Tyr-1173 clone 9H2 (NanoTools, Teningen, Germany); and rabbit polyclonal anti-phospho-Src Tyr-416 (Cell Signaling Technology).

    Techniques: Transfection, Plasmid Preparation, Western Blot

    Reduction of Fhit protein steady-state levels through EGF stimulation in 293 cells. The 293 cells were transiently transfected with EGFR and FHIT expression plasmids and starved for 48 h before stimulation with 100 ng/ml of EGF for the indicated times. Western blot analyses of total lysates were carried out by using Fhit polyclonal antibody (Fhit), a mAb to phosphotyrosine (PY), a mAb to phospho-EGFR Tyr-1173 (P-EGFR Y1173 ), a polyclonal antibody to EGFR (EGFR), and mAb to actin (Actin).

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

    Article Title: FHIT-proteasome degradation caused by mitogenic stimulation of the EGF receptor family in cancer cells

    doi: 10.1073/pnas.0605821103

    Figure Lengend Snippet: Reduction of Fhit protein steady-state levels through EGF stimulation in 293 cells. The 293 cells were transiently transfected with EGFR and FHIT expression plasmids and starved for 48 h before stimulation with 100 ng/ml of EGF for the indicated times. Western blot analyses of total lysates were carried out by using Fhit polyclonal antibody (Fhit), a mAb to phosphotyrosine (PY), a mAb to phospho-EGFR Tyr-1173 (P-EGFR Y1173 ), a polyclonal antibody to EGFR (EGFR), and mAb to actin (Actin).

    Article Snippet: Antibodies used were: mouse monoclonal anti-phosphotyrosine (Upstate Biotechnology, Lake Placid, NY); rabbit polyclonal anti-Fhit (Zymed Laboratories, San Francisco, CA); mouse monoclonal anti-Src clone GD11 (Upstate Biotechnology); mouse monoclonal anti-actin clone AC-40 (Sigma); rabbit polyclonal anti-phospho-Fhit-Tyr-114 (Cell Signaling Technology, Danvers, MA); mouse monoclonal anti-Flag M2 (Sigma); rabbit polyclonal anti-p44/42 MAPK (New England Biolabs, Ipswich, MA); mouse monoclonal anti-phospho p44/42 MAPK (Thr-202/Tyr-204) (Cell Signaling Technology); rabbit polyclonal anti-EGFR clone 1005 (Santa Cruz Biotechnology, Santa Cruz, CA); mouse monoclonal anti-EGFR phospho-Tyr-1173 clone 9H2 (NanoTools, Teningen, Germany); and rabbit polyclonal anti-phospho-Src Tyr-416 (Cell Signaling Technology).

    Techniques: Transfection, Expressing, Western Blot