phospho egfr y845  (New England Biolabs)


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    New England Biolabs phospho egfr y845
    Phospho Egfr Y845, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    rabbit anti phospho egfr y845 polyclonal antibody  (Thermo Fisher)


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    Thermo Fisher rabbit anti phospho egfr y845 polyclonal antibody
    a .Representative patches of MDCK cells under control and <t>EGFR-inhibited</t> conditions (Erlotinib at 1μM) including the tracking or individual cell trajectories. Scale bar: 100 μm. b .Quantification of individual junction elongation velocities in the patches (mean value ± s.d.) n Ctrl = 36 junctions and n Erlotinib = 29 junctions from 3 independent experiments, two-tailed unpaired t-test, p < 0.0001. c .Schematics of the experiment for cell arrest by dextran addition. Quantification of individual cell migration velocity 10 minutes after adding dextran with various molecular weights (mean value ± s.d. n=1735-1925 cells from 3 independent experiments.) d .Western Blot and its quantification of EGFR phosphorylated states <t>(Y845)</t> before and after cell arrest from 4 independent experiments, two-tailed unpaired t-test, p = 0.029. e .Experimental setup schematics (left) and segmented contours quantification (right) of cell mosaically expressing RUSH-EGFR before and after its release from the endoplasmic reticulum. f .Quantifications of junction elongation velocities upon the release of EGFR, under control and pEGFR-inhibited conditions. n Rush/Ctrl = 28 junctions and n Rush/Erlotinib = 15 junctions from 3 independent experiments, two-tailed paired t-test, p Rush/Ctrl < 0.001, p Rush/Erlotinib = 0.52. g .Schematics of the physical induction of cell elongation around obstacles (left). Images of cells encircling obstacles and in bulk regions including single cell tracking and apical localization of pEGFR-Y845 by immunostaining. Scale bar: 50 μm. h .Quantifications of apical pEGFR-Y845 intensity around obstacles (mean value ± s.d. n Bulk = 50 junctions and n Obstacle = 48 junctions from 3 independent experiments, two-tailed unpaired t-test, p<0.0001.) i .Diagram of a positive feedback loop between apical EGFR phosphorylation and cell junction deformation.
    Rabbit Anti Phospho Egfr Y845 Polyclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti phospho egfr y845 polyclonal antibody/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti phospho egfr y845 polyclonal antibody - by Bioz Stars, 2024-07
    86/100 stars

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    1) Product Images from "E-cadherin-dependent phosphorylation of EGFR governs a homeostatic feedback loop controlling intercellular junction viscosity and collective migration modes"

    Article Title: E-cadherin-dependent phosphorylation of EGFR governs a homeostatic feedback loop controlling intercellular junction viscosity and collective migration modes

    Journal: bioRxiv

    doi: 10.1101/2023.12.04.570034

    a .Representative patches of MDCK cells under control and EGFR-inhibited conditions (Erlotinib at 1μM) including the tracking or individual cell trajectories. Scale bar: 100 μm. b .Quantification of individual junction elongation velocities in the patches (mean value ± s.d.) n Ctrl = 36 junctions and n Erlotinib = 29 junctions from 3 independent experiments, two-tailed unpaired t-test, p < 0.0001. c .Schematics of the experiment for cell arrest by dextran addition. Quantification of individual cell migration velocity 10 minutes after adding dextran with various molecular weights (mean value ± s.d. n=1735-1925 cells from 3 independent experiments.) d .Western Blot and its quantification of EGFR phosphorylated states (Y845) before and after cell arrest from 4 independent experiments, two-tailed unpaired t-test, p = 0.029. e .Experimental setup schematics (left) and segmented contours quantification (right) of cell mosaically expressing RUSH-EGFR before and after its release from the endoplasmic reticulum. f .Quantifications of junction elongation velocities upon the release of EGFR, under control and pEGFR-inhibited conditions. n Rush/Ctrl = 28 junctions and n Rush/Erlotinib = 15 junctions from 3 independent experiments, two-tailed paired t-test, p Rush/Ctrl < 0.001, p Rush/Erlotinib = 0.52. g .Schematics of the physical induction of cell elongation around obstacles (left). Images of cells encircling obstacles and in bulk regions including single cell tracking and apical localization of pEGFR-Y845 by immunostaining. Scale bar: 50 μm. h .Quantifications of apical pEGFR-Y845 intensity around obstacles (mean value ± s.d. n Bulk = 50 junctions and n Obstacle = 48 junctions from 3 independent experiments, two-tailed unpaired t-test, p<0.0001.) i .Diagram of a positive feedback loop between apical EGFR phosphorylation and cell junction deformation.
    Figure Legend Snippet: a .Representative patches of MDCK cells under control and EGFR-inhibited conditions (Erlotinib at 1μM) including the tracking or individual cell trajectories. Scale bar: 100 μm. b .Quantification of individual junction elongation velocities in the patches (mean value ± s.d.) n Ctrl = 36 junctions and n Erlotinib = 29 junctions from 3 independent experiments, two-tailed unpaired t-test, p < 0.0001. c .Schematics of the experiment for cell arrest by dextran addition. Quantification of individual cell migration velocity 10 minutes after adding dextran with various molecular weights (mean value ± s.d. n=1735-1925 cells from 3 independent experiments.) d .Western Blot and its quantification of EGFR phosphorylated states (Y845) before and after cell arrest from 4 independent experiments, two-tailed unpaired t-test, p = 0.029. e .Experimental setup schematics (left) and segmented contours quantification (right) of cell mosaically expressing RUSH-EGFR before and after its release from the endoplasmic reticulum. f .Quantifications of junction elongation velocities upon the release of EGFR, under control and pEGFR-inhibited conditions. n Rush/Ctrl = 28 junctions and n Rush/Erlotinib = 15 junctions from 3 independent experiments, two-tailed paired t-test, p Rush/Ctrl < 0.001, p Rush/Erlotinib = 0.52. g .Schematics of the physical induction of cell elongation around obstacles (left). Images of cells encircling obstacles and in bulk regions including single cell tracking and apical localization of pEGFR-Y845 by immunostaining. Scale bar: 50 μm. h .Quantifications of apical pEGFR-Y845 intensity around obstacles (mean value ± s.d. n Bulk = 50 junctions and n Obstacle = 48 junctions from 3 independent experiments, two-tailed unpaired t-test, p<0.0001.) i .Diagram of a positive feedback loop between apical EGFR phosphorylation and cell junction deformation.

    Techniques Used: Two Tailed Test, Migration, Western Blot, Expressing, Single Cell Tracking, Immunostaining

    a .Quantification of cell proliferation (upper left), nucleus area (upper right), circularity (lower left) and aspect ratio (lower right) on cell patches before and after the addition of dextran of the indicated molecular weight. The light blue line indicates the moment of dextran addition. Four independent experiments yielded consistent results. b .Quantification of single-cells velocity and persistence before and after the addition of 270kDa dextran, n Ctrl = 1217 cells and n 270kDa = 543 cells from 3 different experiments, two-tailed unpaired t-test, p<0.0001. c .Representative images of cell tracking with or without the presence of dextran of the indicated molecular weight (up). Quantification of individual cell persistence within cell patches under control or indicated molecular weight dextran conditions (down), n Ctrl = 1762 cells, n 5kDa = 1770 cells, n 50kDa = 1424 cells and n 270kDa = 1968 cells from 3 different experiments, Ordinary one-way ANOVA Tukey’s test, p<0.0001. d .Confocal images (up) and quantification (down) of pEGFR(Y845) junctional fluorescence intensities under control and 270kDa dextran conditions, n Ctrl = 143 cell junctions and n 270kDa = 84 cell junctions from 3 different experiments, two-tailed unpaired t-test, p<0.0001.
    Figure Legend Snippet: a .Quantification of cell proliferation (upper left), nucleus area (upper right), circularity (lower left) and aspect ratio (lower right) on cell patches before and after the addition of dextran of the indicated molecular weight. The light blue line indicates the moment of dextran addition. Four independent experiments yielded consistent results. b .Quantification of single-cells velocity and persistence before and after the addition of 270kDa dextran, n Ctrl = 1217 cells and n 270kDa = 543 cells from 3 different experiments, two-tailed unpaired t-test, p<0.0001. c .Representative images of cell tracking with or without the presence of dextran of the indicated molecular weight (up). Quantification of individual cell persistence within cell patches under control or indicated molecular weight dextran conditions (down), n Ctrl = 1762 cells, n 5kDa = 1770 cells, n 50kDa = 1424 cells and n 270kDa = 1968 cells from 3 different experiments, Ordinary one-way ANOVA Tukey’s test, p<0.0001. d .Confocal images (up) and quantification (down) of pEGFR(Y845) junctional fluorescence intensities under control and 270kDa dextran conditions, n Ctrl = 143 cell junctions and n 270kDa = 84 cell junctions from 3 different experiments, two-tailed unpaired t-test, p<0.0001.

    Techniques Used: Molecular Weight, Two Tailed Test, Cell Tracking Assay, Fluorescence

    a .Immunostaining of apical pEGFR (Y845) and actin in wild-type (WT) and E-cadherin knock-out (Ecad-KO) MDCKs on confluent (C, left) and sub-confluent (SubC, right) regions. Scale Bar: 20 μm. Quantification of apical pEGFR in WT, Ecad-KO and Ecad-KO-rescued (Ecad-Res) tissues. (WT: n C = 122 junctions, n SubC = 106 junctions from 4 independent experiments, p < 0.0001; Ecad-KO: n C = 67 junctions, n SubC = 61 junctions from 3 independent experiments, p = 0.9893; Ecad-Res: n C = 47 junctions, n SubC = 34 cell junctions from 3 independent experiments, p = 0.0239. Ordinary one-way ANOVA Tukey’s test. b .Schematic (left) and time-lapse imaging (middle) of SH2-Grb2 (tdEOS) and E-cadherin (GFP) localization during cell spreading on E-cadherin-coated patterns. Quantification of the recruitment speed of E-cadherin and SH2-Grb2 (right). n Ecad = 40 cell adhesions and n SH2-Grb2 = 132 cell adhesions from 3 independent experiments, two-tailed unpaired t-test, p=0.49. c .Pull-down assays on Rho family GTPases (Rac1, Cdc42 and RhoA) and quantification of GTP-bound GTPases post cell arrest by dextran. n Rac1 = 4 WB, p=0.029, n Cdc42 = 3 WB, p=0.4 and n RhoA = 3 WB, p>1, two-tailed unpaired t-test. d-f . Quantification of apical WAVE2 ( d ), Arp3 ( e ) and pMLC ( f ) under control and EGFR inhibited conditions. Data are the mean value ± s.d. WAVE2: n Ctrl, C = 68 cell junctions, n Ctrl, SubC = 59 cell junctions from 2 independent experiments, n Erlotinib, C = 50 cell junctions, n Erlotinib, SubC = 48 cell junctions from 2 independent experiments; Arp3: n Ctrl, C = 60 cell junctions, n Ctrl, SubC = 43 cell junctions from 2 independent experiments, n Erlotinib, C = 74 cell junctions, n Erlotinib, SubC = 52 cell junctions from 2 independent experiments; pMLC: n Ctrl, C = 49 cell junctions, n Ctrl, SubC = 27 cell junctions from 2 independent experiments, n Erlotinib, C = 62 cell junctions, n Erlotinib, SubC = 39 cell junctions from 2 independent experiments. Ordinary one-way ANOVA Tukey’s test. g . Proposed model of E-cadherin-dependent phosphorylation of EGFR reducing junction viscosity through the regulation of Rac1, WAVE2, Arp2/3, fine-tuning actin dynamics, with minimal impact on cortical tension. h-i . Experimental schematics and characteristic images of fluorescence recovery after photobleaching (FRAP) (left) and laser ablation (right) experiments on intercellular junctions between GFP-Actin-MDCK cells, Scale Bar: 3 μm. Quantification of fluorescence recovery time (left) and recoil velocities (right) under control and pEGFR-inhibited conditions. FRAP: n Ctrl = 36 cell junctions, n Erlotinib = 15 cell junctions from 3 independent experiments, two-tailed unpaired t-test, p<0.0001; Laser ablation: n Ctrl = 10 cell junctions, n Erlotinib = 7 cell junctions from 2 independent experiments, two-tailed unpaired t-test, p=0.8983.
    Figure Legend Snippet: a .Immunostaining of apical pEGFR (Y845) and actin in wild-type (WT) and E-cadherin knock-out (Ecad-KO) MDCKs on confluent (C, left) and sub-confluent (SubC, right) regions. Scale Bar: 20 μm. Quantification of apical pEGFR in WT, Ecad-KO and Ecad-KO-rescued (Ecad-Res) tissues. (WT: n C = 122 junctions, n SubC = 106 junctions from 4 independent experiments, p < 0.0001; Ecad-KO: n C = 67 junctions, n SubC = 61 junctions from 3 independent experiments, p = 0.9893; Ecad-Res: n C = 47 junctions, n SubC = 34 cell junctions from 3 independent experiments, p = 0.0239. Ordinary one-way ANOVA Tukey’s test. b .Schematic (left) and time-lapse imaging (middle) of SH2-Grb2 (tdEOS) and E-cadherin (GFP) localization during cell spreading on E-cadherin-coated patterns. Quantification of the recruitment speed of E-cadherin and SH2-Grb2 (right). n Ecad = 40 cell adhesions and n SH2-Grb2 = 132 cell adhesions from 3 independent experiments, two-tailed unpaired t-test, p=0.49. c .Pull-down assays on Rho family GTPases (Rac1, Cdc42 and RhoA) and quantification of GTP-bound GTPases post cell arrest by dextran. n Rac1 = 4 WB, p=0.029, n Cdc42 = 3 WB, p=0.4 and n RhoA = 3 WB, p>1, two-tailed unpaired t-test. d-f . Quantification of apical WAVE2 ( d ), Arp3 ( e ) and pMLC ( f ) under control and EGFR inhibited conditions. Data are the mean value ± s.d. WAVE2: n Ctrl, C = 68 cell junctions, n Ctrl, SubC = 59 cell junctions from 2 independent experiments, n Erlotinib, C = 50 cell junctions, n Erlotinib, SubC = 48 cell junctions from 2 independent experiments; Arp3: n Ctrl, C = 60 cell junctions, n Ctrl, SubC = 43 cell junctions from 2 independent experiments, n Erlotinib, C = 74 cell junctions, n Erlotinib, SubC = 52 cell junctions from 2 independent experiments; pMLC: n Ctrl, C = 49 cell junctions, n Ctrl, SubC = 27 cell junctions from 2 independent experiments, n Erlotinib, C = 62 cell junctions, n Erlotinib, SubC = 39 cell junctions from 2 independent experiments. Ordinary one-way ANOVA Tukey’s test. g . Proposed model of E-cadherin-dependent phosphorylation of EGFR reducing junction viscosity through the regulation of Rac1, WAVE2, Arp2/3, fine-tuning actin dynamics, with minimal impact on cortical tension. h-i . Experimental schematics and characteristic images of fluorescence recovery after photobleaching (FRAP) (left) and laser ablation (right) experiments on intercellular junctions between GFP-Actin-MDCK cells, Scale Bar: 3 μm. Quantification of fluorescence recovery time (left) and recoil velocities (right) under control and pEGFR-inhibited conditions. FRAP: n Ctrl = 36 cell junctions, n Erlotinib = 15 cell junctions from 3 independent experiments, two-tailed unpaired t-test, p<0.0001; Laser ablation: n Ctrl = 10 cell junctions, n Erlotinib = 7 cell junctions from 2 independent experiments, two-tailed unpaired t-test, p=0.8983.

    Techniques Used: Immunostaining, Knock-Out, Imaging, Two Tailed Test, Viscosity, Fluorescence

    a .Immunostaining of pEGFR (Y845), actin and E-cadherin in wild-type (WT), E-cadherin knock-out (Ecad-KO) and Ecad-KO-rescued (Ecad-Res) MDCK cells on the apical side of confluent (C, left) and sub-confluent (SubC, right) regions under control and pEGFR inhibition (Erlotinib) conditions. The white dotted line indicates the leading front of the patches. Scalr Bar: 20 μm. b .Quantification of apical pEGFR in WT, Ecad-KO and Ecad-Res MDCK cells. For WT: n Ctrl, C = 122 cell junctions, n Ctrl, SubC = 106 cell junctions from 4 different experiments, n Erlotinib, C = 51 cell junctions, n Erlotinib, SubC = 44 cell junctions from 3 different experiments; For Ecad-KO: n Ctrl, C = 67 cell junctions, n Ctrl, SubC = 61 cell junctions from 3 different experiments, n Erlotinib, C = 24 cell junctions, n Erlotinib, SubC = 16 cell junctions from 3 different experiments; For Ecad-Res: n Ctrl, C = 47 cell junctions, n Ctrl, SubC = 34 cell junctions from 3 different experiments, n Erlotinib, C = 41 cell junctions, n Erlotinib, SubC = 32 cell junctions from 3 different experiments, Ordinary one-way ANOVA Tukey’s test. c .RT-qPCR results showing the relative expression of CDH1 (E-cad), CDH2 (N-cad), CDH3 (P-cad) and CDH6 (K-cad) in WT and Ecad-KO MDCK cells from 3 different experiments.
    Figure Legend Snippet: a .Immunostaining of pEGFR (Y845), actin and E-cadherin in wild-type (WT), E-cadherin knock-out (Ecad-KO) and Ecad-KO-rescued (Ecad-Res) MDCK cells on the apical side of confluent (C, left) and sub-confluent (SubC, right) regions under control and pEGFR inhibition (Erlotinib) conditions. The white dotted line indicates the leading front of the patches. Scalr Bar: 20 μm. b .Quantification of apical pEGFR in WT, Ecad-KO and Ecad-Res MDCK cells. For WT: n Ctrl, C = 122 cell junctions, n Ctrl, SubC = 106 cell junctions from 4 different experiments, n Erlotinib, C = 51 cell junctions, n Erlotinib, SubC = 44 cell junctions from 3 different experiments; For Ecad-KO: n Ctrl, C = 67 cell junctions, n Ctrl, SubC = 61 cell junctions from 3 different experiments, n Erlotinib, C = 24 cell junctions, n Erlotinib, SubC = 16 cell junctions from 3 different experiments; For Ecad-Res: n Ctrl, C = 47 cell junctions, n Ctrl, SubC = 34 cell junctions from 3 different experiments, n Erlotinib, C = 41 cell junctions, n Erlotinib, SubC = 32 cell junctions from 3 different experiments, Ordinary one-way ANOVA Tukey’s test. c .RT-qPCR results showing the relative expression of CDH1 (E-cad), CDH2 (N-cad), CDH3 (P-cad) and CDH6 (K-cad) in WT and Ecad-KO MDCK cells from 3 different experiments.

    Techniques Used: Immunostaining, Knock-Out, Inhibition, Quantitative RT-PCR, Expressing

    a . Representative images and corresponding quantification of FRET Ratio in confluent (C, left) and sub-confluent (SubC, right) regions of migrating MDCK monolayers, with or without EGFR inhibition (Erlotinib), n Ctrl, SubC = 27014 cells, n Dextran, SubC = 27766 cells, n Erlotinib, SubC = 15316 cells, n Ctrl, C = 43852 cells, n Dextran, C = 43079 cells and n Erlotinib, C = 27305 cells from 3 independent experiments. Scale Bar: 200 μm. b-c . Immunostaining of WAVE-2 and actin ( b ), Arp3 and actin ( c ) on the apical side of confluent (C, left) and sub-confluent (SubC, right) regions under Control and pEGFR inhibition (Erlotinib) conditions. Scale Bar: 20 μm. Two independent experiments yielded consistent results. d . Immunostaining of pMLC, pEGFR (Y845) and actin on the apical side of confluent (C, left) and sub-confluent (SubC, right) regions under control and pEGFR inhibition (Erlotinib) conditions. Scale Bar: 20 μm. Two independent experiments yielded consistent results.
    Figure Legend Snippet: a . Representative images and corresponding quantification of FRET Ratio in confluent (C, left) and sub-confluent (SubC, right) regions of migrating MDCK monolayers, with or without EGFR inhibition (Erlotinib), n Ctrl, SubC = 27014 cells, n Dextran, SubC = 27766 cells, n Erlotinib, SubC = 15316 cells, n Ctrl, C = 43852 cells, n Dextran, C = 43079 cells and n Erlotinib, C = 27305 cells from 3 independent experiments. Scale Bar: 200 μm. b-c . Immunostaining of WAVE-2 and actin ( b ), Arp3 and actin ( c ) on the apical side of confluent (C, left) and sub-confluent (SubC, right) regions under Control and pEGFR inhibition (Erlotinib) conditions. Scale Bar: 20 μm. Two independent experiments yielded consistent results. d . Immunostaining of pMLC, pEGFR (Y845) and actin on the apical side of confluent (C, left) and sub-confluent (SubC, right) regions under control and pEGFR inhibition (Erlotinib) conditions. Scale Bar: 20 μm. Two independent experiments yielded consistent results.

    Techniques Used: Inhibition, Immunostaining

    a .Phase-contrast images of MDCK monolayers migrating on fibronectin-coated line patterns under control and pEGFR-inhibited conditions (Erlotinib at 1 μM). Scale Bar: 100 μm. Five independent experiments yielded consistent results. b .Immunostaining of apical pEGFR(Y845) highlights its localization at cell junctions in bulk and leading regions under control and leading regions under pEGFR-inhibited conditions. Scale Bar: 20 μm. Four independent experiments corroborate these findings. c-d . Representative velocity ( c ) and vorticity ( d ) profiles with flow line maps, illustrate MDCK monolayer migration under control and pEGFR-inhibited conditions. Scale bar: 100 μm. Three independent experiments yielded consistent results. e .Quantification of cellular strain states in the monolayer under control and pEGFR-inhibited conditions. n Ctrl = 1060 cells and n Erlotinib = 1060 cells from 3 independent experiments, two-tailed unpaired t-test, p<0.0001. f .Quantification of apical localization of pEGFR (Y845) at the bulk and leading front region of the monolayer. n Bulk = 42 cell junctions and n leading = 39 cell junctions from 3 independent experiments, two-tailed unpaired t-test, p < 0.0001. g .Quantification of cell velocity (left) and migration front velocity (right) under control and pEGFR-inhibited conditions. n ctrl, cell = 181 cells, n Erlotinib, cell = 181 cells from 3 independent experiments, p<0.0001; n ctrl, migration front = 16 strips, n Erlotinib, migration front = 12 strips from 3 independent experiments, p=0.45, two-tailed unpaired t-test. h .Quantification of spatial correlation in the velocity field under control and pEGFR-inhibited conditions. n ctrl =181 cells, n Erlotinib =181 cells from 3 independent experiments, two-tailed unpaired t-test, p<0.0001. i .Schematic representation of the analysis pipeline to measure the average cell shape relaxation time in a migrating monolayer. Average cell strain profiles along the migration axis are depicted, with bold lines indicating mean values and narrow lines representing standard deviations. j-k . Correlative plots between measured and advection-based predicted cellular strain for the best fit of the viscoelastic time (t visc ) under control and pEGFR-inhibited conditions. Two independent experiments yielded consistent results. l . Measured viscoelastic time (t visc ) under control and pEGFR-inhibited conditions. n ctrl =3 strips, n Erlotinib =3 strips from 2 independent experiments, two-tailed unpaired t-test, p<0.06.
    Figure Legend Snippet: a .Phase-contrast images of MDCK monolayers migrating on fibronectin-coated line patterns under control and pEGFR-inhibited conditions (Erlotinib at 1 μM). Scale Bar: 100 μm. Five independent experiments yielded consistent results. b .Immunostaining of apical pEGFR(Y845) highlights its localization at cell junctions in bulk and leading regions under control and leading regions under pEGFR-inhibited conditions. Scale Bar: 20 μm. Four independent experiments corroborate these findings. c-d . Representative velocity ( c ) and vorticity ( d ) profiles with flow line maps, illustrate MDCK monolayer migration under control and pEGFR-inhibited conditions. Scale bar: 100 μm. Three independent experiments yielded consistent results. e .Quantification of cellular strain states in the monolayer under control and pEGFR-inhibited conditions. n Ctrl = 1060 cells and n Erlotinib = 1060 cells from 3 independent experiments, two-tailed unpaired t-test, p<0.0001. f .Quantification of apical localization of pEGFR (Y845) at the bulk and leading front region of the monolayer. n Bulk = 42 cell junctions and n leading = 39 cell junctions from 3 independent experiments, two-tailed unpaired t-test, p < 0.0001. g .Quantification of cell velocity (left) and migration front velocity (right) under control and pEGFR-inhibited conditions. n ctrl, cell = 181 cells, n Erlotinib, cell = 181 cells from 3 independent experiments, p<0.0001; n ctrl, migration front = 16 strips, n Erlotinib, migration front = 12 strips from 3 independent experiments, p=0.45, two-tailed unpaired t-test. h .Quantification of spatial correlation in the velocity field under control and pEGFR-inhibited conditions. n ctrl =181 cells, n Erlotinib =181 cells from 3 independent experiments, two-tailed unpaired t-test, p<0.0001. i .Schematic representation of the analysis pipeline to measure the average cell shape relaxation time in a migrating monolayer. Average cell strain profiles along the migration axis are depicted, with bold lines indicating mean values and narrow lines representing standard deviations. j-k . Correlative plots between measured and advection-based predicted cellular strain for the best fit of the viscoelastic time (t visc ) under control and pEGFR-inhibited conditions. Two independent experiments yielded consistent results. l . Measured viscoelastic time (t visc ) under control and pEGFR-inhibited conditions. n ctrl =3 strips, n Erlotinib =3 strips from 2 independent experiments, two-tailed unpaired t-test, p<0.06.

    Techniques Used: Immunostaining, Migration, Two Tailed Test

    anti phospho egfr y845  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc anti phospho egfr y845
    Anti Phospho Egfr Y845, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti phospho egfr y845/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti phospho egfr y845 - by Bioz Stars, 2024-07
    86/100 stars

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    phospho egfr y845  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc phospho egfr y845
    Effect of genistein on expression and phosphorylation of <t>EGFR</t> during experimental CCl 4 -induced subacute liver damage. An increase in total protein and phosphorylation from livers with CCl 4 -induced subacute liver damage was observed. Genistein significantly increased total protein and pY845 and pY1068 EGFR. The analysis was determined by Western blot a semiquantitative analysis of the expression levels of p‑EGFR <t>(Y845,</t> Y992 and Y1068), β-actin was used as a loading control. Bars show the mean values ± standard deviations of the band density normalized to the total protein. n = 8, *p < 0.05 as compared to control group; #p < 0.05 as compared to liver damage group, respectively using an ANOVA and Tukey’s test
    Phospho Egfr Y845, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho egfr y845/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phospho egfr y845 - by Bioz Stars, 2024-07
    86/100 stars

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    1) Product Images from "EGF-receptor phosphorylation and downstream signaling are activated by genistein during subacute liver damage"

    Article Title: EGF-receptor phosphorylation and downstream signaling are activated by genistein during subacute liver damage

    Journal: Journal of Molecular Histology

    doi: 10.1007/s10735-023-10127-8

    Effect of genistein on expression and phosphorylation of EGFR during experimental CCl 4 -induced subacute liver damage. An increase in total protein and phosphorylation from livers with CCl 4 -induced subacute liver damage was observed. Genistein significantly increased total protein and pY845 and pY1068 EGFR. The analysis was determined by Western blot a semiquantitative analysis of the expression levels of p‑EGFR (Y845, Y992 and Y1068), β-actin was used as a loading control. Bars show the mean values ± standard deviations of the band density normalized to the total protein. n = 8, *p < 0.05 as compared to control group; #p < 0.05 as compared to liver damage group, respectively using an ANOVA and Tukey’s test
    Figure Legend Snippet: Effect of genistein on expression and phosphorylation of EGFR during experimental CCl 4 -induced subacute liver damage. An increase in total protein and phosphorylation from livers with CCl 4 -induced subacute liver damage was observed. Genistein significantly increased total protein and pY845 and pY1068 EGFR. The analysis was determined by Western blot a semiquantitative analysis of the expression levels of p‑EGFR (Y845, Y992 and Y1068), β-actin was used as a loading control. Bars show the mean values ± standard deviations of the band density normalized to the total protein. n = 8, *p < 0.05 as compared to control group; #p < 0.05 as compared to liver damage group, respectively using an ANOVA and Tukey’s test

    Techniques Used: Expressing, Western Blot


    Structured Review

    Santa Cruz Biotechnology anti phospho y845 egfr 12a3
    Anti Phospho Y845 Egfr 12a3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti phospho y845 egfr 12a3/product/Santa Cruz Biotechnology
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti phospho y845 egfr 12a3 - by Bioz Stars, 2024-07
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    phospho egfr y845  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc phospho egfr y845
    Phospho Egfr Y845, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho y845 egfr
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    New England Biolabs phospho egfr y845
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    a .Representative patches of MDCK cells under control and <t>EGFR-inhibited</t> conditions (Erlotinib at 1μM) including the tracking or individual cell trajectories. Scale bar: 100 μm. b .Quantification of individual junction elongation velocities in the patches (mean value ± s.d.) n Ctrl = 36 junctions and n Erlotinib = 29 junctions from 3 independent experiments, two-tailed unpaired t-test, p < 0.0001. c .Schematics of the experiment for cell arrest by dextran addition. Quantification of individual cell migration velocity 10 minutes after adding dextran with various molecular weights (mean value ± s.d. n=1735-1925 cells from 3 independent experiments.) d .Western Blot and its quantification of EGFR phosphorylated states <t>(Y845)</t> before and after cell arrest from 4 independent experiments, two-tailed unpaired t-test, p = 0.029. e .Experimental setup schematics (left) and segmented contours quantification (right) of cell mosaically expressing RUSH-EGFR before and after its release from the endoplasmic reticulum. f .Quantifications of junction elongation velocities upon the release of EGFR, under control and pEGFR-inhibited conditions. n Rush/Ctrl = 28 junctions and n Rush/Erlotinib = 15 junctions from 3 independent experiments, two-tailed paired t-test, p Rush/Ctrl < 0.001, p Rush/Erlotinib = 0.52. g .Schematics of the physical induction of cell elongation around obstacles (left). Images of cells encircling obstacles and in bulk regions including single cell tracking and apical localization of pEGFR-Y845 by immunostaining. Scale bar: 50 μm. h .Quantifications of apical pEGFR-Y845 intensity around obstacles (mean value ± s.d. n Bulk = 50 junctions and n Obstacle = 48 junctions from 3 independent experiments, two-tailed unpaired t-test, p<0.0001.) i .Diagram of a positive feedback loop between apical EGFR phosphorylation and cell junction deformation.
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    a .Representative patches of MDCK cells under control and <t>EGFR-inhibited</t> conditions (Erlotinib at 1μM) including the tracking or individual cell trajectories. Scale bar: 100 μm. b .Quantification of individual junction elongation velocities in the patches (mean value ± s.d.) n Ctrl = 36 junctions and n Erlotinib = 29 junctions from 3 independent experiments, two-tailed unpaired t-test, p < 0.0001. c .Schematics of the experiment for cell arrest by dextran addition. Quantification of individual cell migration velocity 10 minutes after adding dextran with various molecular weights (mean value ± s.d. n=1735-1925 cells from 3 independent experiments.) d .Western Blot and its quantification of EGFR phosphorylated states <t>(Y845)</t> before and after cell arrest from 4 independent experiments, two-tailed unpaired t-test, p = 0.029. e .Experimental setup schematics (left) and segmented contours quantification (right) of cell mosaically expressing RUSH-EGFR before and after its release from the endoplasmic reticulum. f .Quantifications of junction elongation velocities upon the release of EGFR, under control and pEGFR-inhibited conditions. n Rush/Ctrl = 28 junctions and n Rush/Erlotinib = 15 junctions from 3 independent experiments, two-tailed paired t-test, p Rush/Ctrl < 0.001, p Rush/Erlotinib = 0.52. g .Schematics of the physical induction of cell elongation around obstacles (left). Images of cells encircling obstacles and in bulk regions including single cell tracking and apical localization of pEGFR-Y845 by immunostaining. Scale bar: 50 μm. h .Quantifications of apical pEGFR-Y845 intensity around obstacles (mean value ± s.d. n Bulk = 50 junctions and n Obstacle = 48 junctions from 3 independent experiments, two-tailed unpaired t-test, p<0.0001.) i .Diagram of a positive feedback loop between apical EGFR phosphorylation and cell junction deformation.
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    Effect of genistein on expression and phosphorylation of <t>EGFR</t> during experimental CCl 4 -induced subacute liver damage. An increase in total protein and phosphorylation from livers with CCl 4 -induced subacute liver damage was observed. Genistein significantly increased total protein and pY845 and pY1068 EGFR. The analysis was determined by Western blot a semiquantitative analysis of the expression levels of p‑EGFR <t>(Y845,</t> Y992 and Y1068), β-actin was used as a loading control. Bars show the mean values ± standard deviations of the band density normalized to the total protein. n = 8, *p < 0.05 as compared to control group; #p < 0.05 as compared to liver damage group, respectively using an ANOVA and Tukey’s test
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    Effect of genistein on expression and phosphorylation of <t>EGFR</t> during experimental CCl 4 -induced subacute liver damage. An increase in total protein and phosphorylation from livers with CCl 4 -induced subacute liver damage was observed. Genistein significantly increased total protein and pY845 and pY1068 EGFR. The analysis was determined by Western blot a semiquantitative analysis of the expression levels of p‑EGFR <t>(Y845,</t> Y992 and Y1068), β-actin was used as a loading control. Bars show the mean values ± standard deviations of the band density normalized to the total protein. n = 8, *p < 0.05 as compared to control group; #p < 0.05 as compared to liver damage group, respectively using an ANOVA and Tukey’s test
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    Effect of genistein on expression and phosphorylation of <t>EGFR</t> during experimental CCl 4 -induced subacute liver damage. An increase in total protein and phosphorylation from livers with CCl 4 -induced subacute liver damage was observed. Genistein significantly increased total protein and pY845 and pY1068 EGFR. The analysis was determined by Western blot a semiquantitative analysis of the expression levels of p‑EGFR <t>(Y845,</t> Y992 and Y1068), β-actin was used as a loading control. Bars show the mean values ± standard deviations of the band density normalized to the total protein. n = 8, *p < 0.05 as compared to control group; #p < 0.05 as compared to liver damage group, respectively using an ANOVA and Tukey’s test
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    Effect of genistein on expression and phosphorylation of <t>EGFR</t> during experimental CCl 4 -induced subacute liver damage. An increase in total protein and phosphorylation from livers with CCl 4 -induced subacute liver damage was observed. Genistein significantly increased total protein and pY845 and pY1068 EGFR. The analysis was determined by Western blot a semiquantitative analysis of the expression levels of p‑EGFR <t>(Y845,</t> Y992 and Y1068), β-actin was used as a loading control. Bars show the mean values ± standard deviations of the band density normalized to the total protein. n = 8, *p < 0.05 as compared to control group; #p < 0.05 as compared to liver damage group, respectively using an ANOVA and Tukey’s test
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    Image Search Results


    a .Representative patches of MDCK cells under control and EGFR-inhibited conditions (Erlotinib at 1μM) including the tracking or individual cell trajectories. Scale bar: 100 μm. b .Quantification of individual junction elongation velocities in the patches (mean value ± s.d.) n Ctrl = 36 junctions and n Erlotinib = 29 junctions from 3 independent experiments, two-tailed unpaired t-test, p < 0.0001. c .Schematics of the experiment for cell arrest by dextran addition. Quantification of individual cell migration velocity 10 minutes after adding dextran with various molecular weights (mean value ± s.d. n=1735-1925 cells from 3 independent experiments.) d .Western Blot and its quantification of EGFR phosphorylated states (Y845) before and after cell arrest from 4 independent experiments, two-tailed unpaired t-test, p = 0.029. e .Experimental setup schematics (left) and segmented contours quantification (right) of cell mosaically expressing RUSH-EGFR before and after its release from the endoplasmic reticulum. f .Quantifications of junction elongation velocities upon the release of EGFR, under control and pEGFR-inhibited conditions. n Rush/Ctrl = 28 junctions and n Rush/Erlotinib = 15 junctions from 3 independent experiments, two-tailed paired t-test, p Rush/Ctrl < 0.001, p Rush/Erlotinib = 0.52. g .Schematics of the physical induction of cell elongation around obstacles (left). Images of cells encircling obstacles and in bulk regions including single cell tracking and apical localization of pEGFR-Y845 by immunostaining. Scale bar: 50 μm. h .Quantifications of apical pEGFR-Y845 intensity around obstacles (mean value ± s.d. n Bulk = 50 junctions and n Obstacle = 48 junctions from 3 independent experiments, two-tailed unpaired t-test, p<0.0001.) i .Diagram of a positive feedback loop between apical EGFR phosphorylation and cell junction deformation.

    Journal: bioRxiv

    Article Title: E-cadherin-dependent phosphorylation of EGFR governs a homeostatic feedback loop controlling intercellular junction viscosity and collective migration modes

    doi: 10.1101/2023.12.04.570034

    Figure Lengend Snippet: a .Representative patches of MDCK cells under control and EGFR-inhibited conditions (Erlotinib at 1μM) including the tracking or individual cell trajectories. Scale bar: 100 μm. b .Quantification of individual junction elongation velocities in the patches (mean value ± s.d.) n Ctrl = 36 junctions and n Erlotinib = 29 junctions from 3 independent experiments, two-tailed unpaired t-test, p < 0.0001. c .Schematics of the experiment for cell arrest by dextran addition. Quantification of individual cell migration velocity 10 minutes after adding dextran with various molecular weights (mean value ± s.d. n=1735-1925 cells from 3 independent experiments.) d .Western Blot and its quantification of EGFR phosphorylated states (Y845) before and after cell arrest from 4 independent experiments, two-tailed unpaired t-test, p = 0.029. e .Experimental setup schematics (left) and segmented contours quantification (right) of cell mosaically expressing RUSH-EGFR before and after its release from the endoplasmic reticulum. f .Quantifications of junction elongation velocities upon the release of EGFR, under control and pEGFR-inhibited conditions. n Rush/Ctrl = 28 junctions and n Rush/Erlotinib = 15 junctions from 3 independent experiments, two-tailed paired t-test, p Rush/Ctrl < 0.001, p Rush/Erlotinib = 0.52. g .Schematics of the physical induction of cell elongation around obstacles (left). Images of cells encircling obstacles and in bulk regions including single cell tracking and apical localization of pEGFR-Y845 by immunostaining. Scale bar: 50 μm. h .Quantifications of apical pEGFR-Y845 intensity around obstacles (mean value ± s.d. n Bulk = 50 junctions and n Obstacle = 48 junctions from 3 independent experiments, two-tailed unpaired t-test, p<0.0001.) i .Diagram of a positive feedback loop between apical EGFR phosphorylation and cell junction deformation.

    Article Snippet: The cells were incubated overnight at 4 °C with primary antibodies: rabbit anti-phospho-EGFR (Y845) polyclonal antibody (44-784G, Thermo Fisher Scientific, diluted 1:200); Purified Mouse anti-E-Cadherin monoclonal antibody (Clone 36) (610181, BD Transduction Laboratories); WAVE2 antibody (H-110) (sc-33548, Santa Cruz); Arp3 antibody (A5979, Sigma-Aldrich); Phospho-Myosin Light Chain 2 (Ser19) antibody (3671, Cell Signaling Technologies, diluted 1:50) according to the manufacturer’s instructions.

    Techniques: Two Tailed Test, Migration, Western Blot, Expressing, Single Cell Tracking, Immunostaining

    a .Quantification of cell proliferation (upper left), nucleus area (upper right), circularity (lower left) and aspect ratio (lower right) on cell patches before and after the addition of dextran of the indicated molecular weight. The light blue line indicates the moment of dextran addition. Four independent experiments yielded consistent results. b .Quantification of single-cells velocity and persistence before and after the addition of 270kDa dextran, n Ctrl = 1217 cells and n 270kDa = 543 cells from 3 different experiments, two-tailed unpaired t-test, p<0.0001. c .Representative images of cell tracking with or without the presence of dextran of the indicated molecular weight (up). Quantification of individual cell persistence within cell patches under control or indicated molecular weight dextran conditions (down), n Ctrl = 1762 cells, n 5kDa = 1770 cells, n 50kDa = 1424 cells and n 270kDa = 1968 cells from 3 different experiments, Ordinary one-way ANOVA Tukey’s test, p<0.0001. d .Confocal images (up) and quantification (down) of pEGFR(Y845) junctional fluorescence intensities under control and 270kDa dextran conditions, n Ctrl = 143 cell junctions and n 270kDa = 84 cell junctions from 3 different experiments, two-tailed unpaired t-test, p<0.0001.

    Journal: bioRxiv

    Article Title: E-cadherin-dependent phosphorylation of EGFR governs a homeostatic feedback loop controlling intercellular junction viscosity and collective migration modes

    doi: 10.1101/2023.12.04.570034

    Figure Lengend Snippet: a .Quantification of cell proliferation (upper left), nucleus area (upper right), circularity (lower left) and aspect ratio (lower right) on cell patches before and after the addition of dextran of the indicated molecular weight. The light blue line indicates the moment of dextran addition. Four independent experiments yielded consistent results. b .Quantification of single-cells velocity and persistence before and after the addition of 270kDa dextran, n Ctrl = 1217 cells and n 270kDa = 543 cells from 3 different experiments, two-tailed unpaired t-test, p<0.0001. c .Representative images of cell tracking with or without the presence of dextran of the indicated molecular weight (up). Quantification of individual cell persistence within cell patches under control or indicated molecular weight dextran conditions (down), n Ctrl = 1762 cells, n 5kDa = 1770 cells, n 50kDa = 1424 cells and n 270kDa = 1968 cells from 3 different experiments, Ordinary one-way ANOVA Tukey’s test, p<0.0001. d .Confocal images (up) and quantification (down) of pEGFR(Y845) junctional fluorescence intensities under control and 270kDa dextran conditions, n Ctrl = 143 cell junctions and n 270kDa = 84 cell junctions from 3 different experiments, two-tailed unpaired t-test, p<0.0001.

    Article Snippet: The cells were incubated overnight at 4 °C with primary antibodies: rabbit anti-phospho-EGFR (Y845) polyclonal antibody (44-784G, Thermo Fisher Scientific, diluted 1:200); Purified Mouse anti-E-Cadherin monoclonal antibody (Clone 36) (610181, BD Transduction Laboratories); WAVE2 antibody (H-110) (sc-33548, Santa Cruz); Arp3 antibody (A5979, Sigma-Aldrich); Phospho-Myosin Light Chain 2 (Ser19) antibody (3671, Cell Signaling Technologies, diluted 1:50) according to the manufacturer’s instructions.

    Techniques: Molecular Weight, Two Tailed Test, Cell Tracking Assay, Fluorescence

    a .Immunostaining of apical pEGFR (Y845) and actin in wild-type (WT) and E-cadherin knock-out (Ecad-KO) MDCKs on confluent (C, left) and sub-confluent (SubC, right) regions. Scale Bar: 20 μm. Quantification of apical pEGFR in WT, Ecad-KO and Ecad-KO-rescued (Ecad-Res) tissues. (WT: n C = 122 junctions, n SubC = 106 junctions from 4 independent experiments, p < 0.0001; Ecad-KO: n C = 67 junctions, n SubC = 61 junctions from 3 independent experiments, p = 0.9893; Ecad-Res: n C = 47 junctions, n SubC = 34 cell junctions from 3 independent experiments, p = 0.0239. Ordinary one-way ANOVA Tukey’s test. b .Schematic (left) and time-lapse imaging (middle) of SH2-Grb2 (tdEOS) and E-cadherin (GFP) localization during cell spreading on E-cadherin-coated patterns. Quantification of the recruitment speed of E-cadherin and SH2-Grb2 (right). n Ecad = 40 cell adhesions and n SH2-Grb2 = 132 cell adhesions from 3 independent experiments, two-tailed unpaired t-test, p=0.49. c .Pull-down assays on Rho family GTPases (Rac1, Cdc42 and RhoA) and quantification of GTP-bound GTPases post cell arrest by dextran. n Rac1 = 4 WB, p=0.029, n Cdc42 = 3 WB, p=0.4 and n RhoA = 3 WB, p>1, two-tailed unpaired t-test. d-f . Quantification of apical WAVE2 ( d ), Arp3 ( e ) and pMLC ( f ) under control and EGFR inhibited conditions. Data are the mean value ± s.d. WAVE2: n Ctrl, C = 68 cell junctions, n Ctrl, SubC = 59 cell junctions from 2 independent experiments, n Erlotinib, C = 50 cell junctions, n Erlotinib, SubC = 48 cell junctions from 2 independent experiments; Arp3: n Ctrl, C = 60 cell junctions, n Ctrl, SubC = 43 cell junctions from 2 independent experiments, n Erlotinib, C = 74 cell junctions, n Erlotinib, SubC = 52 cell junctions from 2 independent experiments; pMLC: n Ctrl, C = 49 cell junctions, n Ctrl, SubC = 27 cell junctions from 2 independent experiments, n Erlotinib, C = 62 cell junctions, n Erlotinib, SubC = 39 cell junctions from 2 independent experiments. Ordinary one-way ANOVA Tukey’s test. g . Proposed model of E-cadherin-dependent phosphorylation of EGFR reducing junction viscosity through the regulation of Rac1, WAVE2, Arp2/3, fine-tuning actin dynamics, with minimal impact on cortical tension. h-i . Experimental schematics and characteristic images of fluorescence recovery after photobleaching (FRAP) (left) and laser ablation (right) experiments on intercellular junctions between GFP-Actin-MDCK cells, Scale Bar: 3 μm. Quantification of fluorescence recovery time (left) and recoil velocities (right) under control and pEGFR-inhibited conditions. FRAP: n Ctrl = 36 cell junctions, n Erlotinib = 15 cell junctions from 3 independent experiments, two-tailed unpaired t-test, p<0.0001; Laser ablation: n Ctrl = 10 cell junctions, n Erlotinib = 7 cell junctions from 2 independent experiments, two-tailed unpaired t-test, p=0.8983.

    Journal: bioRxiv

    Article Title: E-cadherin-dependent phosphorylation of EGFR governs a homeostatic feedback loop controlling intercellular junction viscosity and collective migration modes

    doi: 10.1101/2023.12.04.570034

    Figure Lengend Snippet: a .Immunostaining of apical pEGFR (Y845) and actin in wild-type (WT) and E-cadherin knock-out (Ecad-KO) MDCKs on confluent (C, left) and sub-confluent (SubC, right) regions. Scale Bar: 20 μm. Quantification of apical pEGFR in WT, Ecad-KO and Ecad-KO-rescued (Ecad-Res) tissues. (WT: n C = 122 junctions, n SubC = 106 junctions from 4 independent experiments, p < 0.0001; Ecad-KO: n C = 67 junctions, n SubC = 61 junctions from 3 independent experiments, p = 0.9893; Ecad-Res: n C = 47 junctions, n SubC = 34 cell junctions from 3 independent experiments, p = 0.0239. Ordinary one-way ANOVA Tukey’s test. b .Schematic (left) and time-lapse imaging (middle) of SH2-Grb2 (tdEOS) and E-cadherin (GFP) localization during cell spreading on E-cadherin-coated patterns. Quantification of the recruitment speed of E-cadherin and SH2-Grb2 (right). n Ecad = 40 cell adhesions and n SH2-Grb2 = 132 cell adhesions from 3 independent experiments, two-tailed unpaired t-test, p=0.49. c .Pull-down assays on Rho family GTPases (Rac1, Cdc42 and RhoA) and quantification of GTP-bound GTPases post cell arrest by dextran. n Rac1 = 4 WB, p=0.029, n Cdc42 = 3 WB, p=0.4 and n RhoA = 3 WB, p>1, two-tailed unpaired t-test. d-f . Quantification of apical WAVE2 ( d ), Arp3 ( e ) and pMLC ( f ) under control and EGFR inhibited conditions. Data are the mean value ± s.d. WAVE2: n Ctrl, C = 68 cell junctions, n Ctrl, SubC = 59 cell junctions from 2 independent experiments, n Erlotinib, C = 50 cell junctions, n Erlotinib, SubC = 48 cell junctions from 2 independent experiments; Arp3: n Ctrl, C = 60 cell junctions, n Ctrl, SubC = 43 cell junctions from 2 independent experiments, n Erlotinib, C = 74 cell junctions, n Erlotinib, SubC = 52 cell junctions from 2 independent experiments; pMLC: n Ctrl, C = 49 cell junctions, n Ctrl, SubC = 27 cell junctions from 2 independent experiments, n Erlotinib, C = 62 cell junctions, n Erlotinib, SubC = 39 cell junctions from 2 independent experiments. Ordinary one-way ANOVA Tukey’s test. g . Proposed model of E-cadherin-dependent phosphorylation of EGFR reducing junction viscosity through the regulation of Rac1, WAVE2, Arp2/3, fine-tuning actin dynamics, with minimal impact on cortical tension. h-i . Experimental schematics and characteristic images of fluorescence recovery after photobleaching (FRAP) (left) and laser ablation (right) experiments on intercellular junctions between GFP-Actin-MDCK cells, Scale Bar: 3 μm. Quantification of fluorescence recovery time (left) and recoil velocities (right) under control and pEGFR-inhibited conditions. FRAP: n Ctrl = 36 cell junctions, n Erlotinib = 15 cell junctions from 3 independent experiments, two-tailed unpaired t-test, p<0.0001; Laser ablation: n Ctrl = 10 cell junctions, n Erlotinib = 7 cell junctions from 2 independent experiments, two-tailed unpaired t-test, p=0.8983.

    Article Snippet: The cells were incubated overnight at 4 °C with primary antibodies: rabbit anti-phospho-EGFR (Y845) polyclonal antibody (44-784G, Thermo Fisher Scientific, diluted 1:200); Purified Mouse anti-E-Cadherin monoclonal antibody (Clone 36) (610181, BD Transduction Laboratories); WAVE2 antibody (H-110) (sc-33548, Santa Cruz); Arp3 antibody (A5979, Sigma-Aldrich); Phospho-Myosin Light Chain 2 (Ser19) antibody (3671, Cell Signaling Technologies, diluted 1:50) according to the manufacturer’s instructions.

    Techniques: Immunostaining, Knock-Out, Imaging, Two Tailed Test, Viscosity, Fluorescence

    a .Immunostaining of pEGFR (Y845), actin and E-cadherin in wild-type (WT), E-cadherin knock-out (Ecad-KO) and Ecad-KO-rescued (Ecad-Res) MDCK cells on the apical side of confluent (C, left) and sub-confluent (SubC, right) regions under control and pEGFR inhibition (Erlotinib) conditions. The white dotted line indicates the leading front of the patches. Scalr Bar: 20 μm. b .Quantification of apical pEGFR in WT, Ecad-KO and Ecad-Res MDCK cells. For WT: n Ctrl, C = 122 cell junctions, n Ctrl, SubC = 106 cell junctions from 4 different experiments, n Erlotinib, C = 51 cell junctions, n Erlotinib, SubC = 44 cell junctions from 3 different experiments; For Ecad-KO: n Ctrl, C = 67 cell junctions, n Ctrl, SubC = 61 cell junctions from 3 different experiments, n Erlotinib, C = 24 cell junctions, n Erlotinib, SubC = 16 cell junctions from 3 different experiments; For Ecad-Res: n Ctrl, C = 47 cell junctions, n Ctrl, SubC = 34 cell junctions from 3 different experiments, n Erlotinib, C = 41 cell junctions, n Erlotinib, SubC = 32 cell junctions from 3 different experiments, Ordinary one-way ANOVA Tukey’s test. c .RT-qPCR results showing the relative expression of CDH1 (E-cad), CDH2 (N-cad), CDH3 (P-cad) and CDH6 (K-cad) in WT and Ecad-KO MDCK cells from 3 different experiments.

    Journal: bioRxiv

    Article Title: E-cadherin-dependent phosphorylation of EGFR governs a homeostatic feedback loop controlling intercellular junction viscosity and collective migration modes

    doi: 10.1101/2023.12.04.570034

    Figure Lengend Snippet: a .Immunostaining of pEGFR (Y845), actin and E-cadherin in wild-type (WT), E-cadherin knock-out (Ecad-KO) and Ecad-KO-rescued (Ecad-Res) MDCK cells on the apical side of confluent (C, left) and sub-confluent (SubC, right) regions under control and pEGFR inhibition (Erlotinib) conditions. The white dotted line indicates the leading front of the patches. Scalr Bar: 20 μm. b .Quantification of apical pEGFR in WT, Ecad-KO and Ecad-Res MDCK cells. For WT: n Ctrl, C = 122 cell junctions, n Ctrl, SubC = 106 cell junctions from 4 different experiments, n Erlotinib, C = 51 cell junctions, n Erlotinib, SubC = 44 cell junctions from 3 different experiments; For Ecad-KO: n Ctrl, C = 67 cell junctions, n Ctrl, SubC = 61 cell junctions from 3 different experiments, n Erlotinib, C = 24 cell junctions, n Erlotinib, SubC = 16 cell junctions from 3 different experiments; For Ecad-Res: n Ctrl, C = 47 cell junctions, n Ctrl, SubC = 34 cell junctions from 3 different experiments, n Erlotinib, C = 41 cell junctions, n Erlotinib, SubC = 32 cell junctions from 3 different experiments, Ordinary one-way ANOVA Tukey’s test. c .RT-qPCR results showing the relative expression of CDH1 (E-cad), CDH2 (N-cad), CDH3 (P-cad) and CDH6 (K-cad) in WT and Ecad-KO MDCK cells from 3 different experiments.

    Article Snippet: The cells were incubated overnight at 4 °C with primary antibodies: rabbit anti-phospho-EGFR (Y845) polyclonal antibody (44-784G, Thermo Fisher Scientific, diluted 1:200); Purified Mouse anti-E-Cadherin monoclonal antibody (Clone 36) (610181, BD Transduction Laboratories); WAVE2 antibody (H-110) (sc-33548, Santa Cruz); Arp3 antibody (A5979, Sigma-Aldrich); Phospho-Myosin Light Chain 2 (Ser19) antibody (3671, Cell Signaling Technologies, diluted 1:50) according to the manufacturer’s instructions.

    Techniques: Immunostaining, Knock-Out, Inhibition, Quantitative RT-PCR, Expressing

    a . Representative images and corresponding quantification of FRET Ratio in confluent (C, left) and sub-confluent (SubC, right) regions of migrating MDCK monolayers, with or without EGFR inhibition (Erlotinib), n Ctrl, SubC = 27014 cells, n Dextran, SubC = 27766 cells, n Erlotinib, SubC = 15316 cells, n Ctrl, C = 43852 cells, n Dextran, C = 43079 cells and n Erlotinib, C = 27305 cells from 3 independent experiments. Scale Bar: 200 μm. b-c . Immunostaining of WAVE-2 and actin ( b ), Arp3 and actin ( c ) on the apical side of confluent (C, left) and sub-confluent (SubC, right) regions under Control and pEGFR inhibition (Erlotinib) conditions. Scale Bar: 20 μm. Two independent experiments yielded consistent results. d . Immunostaining of pMLC, pEGFR (Y845) and actin on the apical side of confluent (C, left) and sub-confluent (SubC, right) regions under control and pEGFR inhibition (Erlotinib) conditions. Scale Bar: 20 μm. Two independent experiments yielded consistent results.

    Journal: bioRxiv

    Article Title: E-cadherin-dependent phosphorylation of EGFR governs a homeostatic feedback loop controlling intercellular junction viscosity and collective migration modes

    doi: 10.1101/2023.12.04.570034

    Figure Lengend Snippet: a . Representative images and corresponding quantification of FRET Ratio in confluent (C, left) and sub-confluent (SubC, right) regions of migrating MDCK monolayers, with or without EGFR inhibition (Erlotinib), n Ctrl, SubC = 27014 cells, n Dextran, SubC = 27766 cells, n Erlotinib, SubC = 15316 cells, n Ctrl, C = 43852 cells, n Dextran, C = 43079 cells and n Erlotinib, C = 27305 cells from 3 independent experiments. Scale Bar: 200 μm. b-c . Immunostaining of WAVE-2 and actin ( b ), Arp3 and actin ( c ) on the apical side of confluent (C, left) and sub-confluent (SubC, right) regions under Control and pEGFR inhibition (Erlotinib) conditions. Scale Bar: 20 μm. Two independent experiments yielded consistent results. d . Immunostaining of pMLC, pEGFR (Y845) and actin on the apical side of confluent (C, left) and sub-confluent (SubC, right) regions under control and pEGFR inhibition (Erlotinib) conditions. Scale Bar: 20 μm. Two independent experiments yielded consistent results.

    Article Snippet: The cells were incubated overnight at 4 °C with primary antibodies: rabbit anti-phospho-EGFR (Y845) polyclonal antibody (44-784G, Thermo Fisher Scientific, diluted 1:200); Purified Mouse anti-E-Cadherin monoclonal antibody (Clone 36) (610181, BD Transduction Laboratories); WAVE2 antibody (H-110) (sc-33548, Santa Cruz); Arp3 antibody (A5979, Sigma-Aldrich); Phospho-Myosin Light Chain 2 (Ser19) antibody (3671, Cell Signaling Technologies, diluted 1:50) according to the manufacturer’s instructions.

    Techniques: Inhibition, Immunostaining

    a .Phase-contrast images of MDCK monolayers migrating on fibronectin-coated line patterns under control and pEGFR-inhibited conditions (Erlotinib at 1 μM). Scale Bar: 100 μm. Five independent experiments yielded consistent results. b .Immunostaining of apical pEGFR(Y845) highlights its localization at cell junctions in bulk and leading regions under control and leading regions under pEGFR-inhibited conditions. Scale Bar: 20 μm. Four independent experiments corroborate these findings. c-d . Representative velocity ( c ) and vorticity ( d ) profiles with flow line maps, illustrate MDCK monolayer migration under control and pEGFR-inhibited conditions. Scale bar: 100 μm. Three independent experiments yielded consistent results. e .Quantification of cellular strain states in the monolayer under control and pEGFR-inhibited conditions. n Ctrl = 1060 cells and n Erlotinib = 1060 cells from 3 independent experiments, two-tailed unpaired t-test, p<0.0001. f .Quantification of apical localization of pEGFR (Y845) at the bulk and leading front region of the monolayer. n Bulk = 42 cell junctions and n leading = 39 cell junctions from 3 independent experiments, two-tailed unpaired t-test, p < 0.0001. g .Quantification of cell velocity (left) and migration front velocity (right) under control and pEGFR-inhibited conditions. n ctrl, cell = 181 cells, n Erlotinib, cell = 181 cells from 3 independent experiments, p<0.0001; n ctrl, migration front = 16 strips, n Erlotinib, migration front = 12 strips from 3 independent experiments, p=0.45, two-tailed unpaired t-test. h .Quantification of spatial correlation in the velocity field under control and pEGFR-inhibited conditions. n ctrl =181 cells, n Erlotinib =181 cells from 3 independent experiments, two-tailed unpaired t-test, p<0.0001. i .Schematic representation of the analysis pipeline to measure the average cell shape relaxation time in a migrating monolayer. Average cell strain profiles along the migration axis are depicted, with bold lines indicating mean values and narrow lines representing standard deviations. j-k . Correlative plots between measured and advection-based predicted cellular strain for the best fit of the viscoelastic time (t visc ) under control and pEGFR-inhibited conditions. Two independent experiments yielded consistent results. l . Measured viscoelastic time (t visc ) under control and pEGFR-inhibited conditions. n ctrl =3 strips, n Erlotinib =3 strips from 2 independent experiments, two-tailed unpaired t-test, p<0.06.

    Journal: bioRxiv

    Article Title: E-cadherin-dependent phosphorylation of EGFR governs a homeostatic feedback loop controlling intercellular junction viscosity and collective migration modes

    doi: 10.1101/2023.12.04.570034

    Figure Lengend Snippet: a .Phase-contrast images of MDCK monolayers migrating on fibronectin-coated line patterns under control and pEGFR-inhibited conditions (Erlotinib at 1 μM). Scale Bar: 100 μm. Five independent experiments yielded consistent results. b .Immunostaining of apical pEGFR(Y845) highlights its localization at cell junctions in bulk and leading regions under control and leading regions under pEGFR-inhibited conditions. Scale Bar: 20 μm. Four independent experiments corroborate these findings. c-d . Representative velocity ( c ) and vorticity ( d ) profiles with flow line maps, illustrate MDCK monolayer migration under control and pEGFR-inhibited conditions. Scale bar: 100 μm. Three independent experiments yielded consistent results. e .Quantification of cellular strain states in the monolayer under control and pEGFR-inhibited conditions. n Ctrl = 1060 cells and n Erlotinib = 1060 cells from 3 independent experiments, two-tailed unpaired t-test, p<0.0001. f .Quantification of apical localization of pEGFR (Y845) at the bulk and leading front region of the monolayer. n Bulk = 42 cell junctions and n leading = 39 cell junctions from 3 independent experiments, two-tailed unpaired t-test, p < 0.0001. g .Quantification of cell velocity (left) and migration front velocity (right) under control and pEGFR-inhibited conditions. n ctrl, cell = 181 cells, n Erlotinib, cell = 181 cells from 3 independent experiments, p<0.0001; n ctrl, migration front = 16 strips, n Erlotinib, migration front = 12 strips from 3 independent experiments, p=0.45, two-tailed unpaired t-test. h .Quantification of spatial correlation in the velocity field under control and pEGFR-inhibited conditions. n ctrl =181 cells, n Erlotinib =181 cells from 3 independent experiments, two-tailed unpaired t-test, p<0.0001. i .Schematic representation of the analysis pipeline to measure the average cell shape relaxation time in a migrating monolayer. Average cell strain profiles along the migration axis are depicted, with bold lines indicating mean values and narrow lines representing standard deviations. j-k . Correlative plots between measured and advection-based predicted cellular strain for the best fit of the viscoelastic time (t visc ) under control and pEGFR-inhibited conditions. Two independent experiments yielded consistent results. l . Measured viscoelastic time (t visc ) under control and pEGFR-inhibited conditions. n ctrl =3 strips, n Erlotinib =3 strips from 2 independent experiments, two-tailed unpaired t-test, p<0.06.

    Article Snippet: The cells were incubated overnight at 4 °C with primary antibodies: rabbit anti-phospho-EGFR (Y845) polyclonal antibody (44-784G, Thermo Fisher Scientific, diluted 1:200); Purified Mouse anti-E-Cadherin monoclonal antibody (Clone 36) (610181, BD Transduction Laboratories); WAVE2 antibody (H-110) (sc-33548, Santa Cruz); Arp3 antibody (A5979, Sigma-Aldrich); Phospho-Myosin Light Chain 2 (Ser19) antibody (3671, Cell Signaling Technologies, diluted 1:50) according to the manufacturer’s instructions.

    Techniques: Immunostaining, Migration, Two Tailed Test

    Effect of genistein on expression and phosphorylation of EGFR during experimental CCl 4 -induced subacute liver damage. An increase in total protein and phosphorylation from livers with CCl 4 -induced subacute liver damage was observed. Genistein significantly increased total protein and pY845 and pY1068 EGFR. The analysis was determined by Western blot a semiquantitative analysis of the expression levels of p‑EGFR (Y845, Y992 and Y1068), β-actin was used as a loading control. Bars show the mean values ± standard deviations of the band density normalized to the total protein. n = 8, *p < 0.05 as compared to control group; #p < 0.05 as compared to liver damage group, respectively using an ANOVA and Tukey’s test

    Journal: Journal of Molecular Histology

    Article Title: EGF-receptor phosphorylation and downstream signaling are activated by genistein during subacute liver damage

    doi: 10.1007/s10735-023-10127-8

    Figure Lengend Snippet: Effect of genistein on expression and phosphorylation of EGFR during experimental CCl 4 -induced subacute liver damage. An increase in total protein and phosphorylation from livers with CCl 4 -induced subacute liver damage was observed. Genistein significantly increased total protein and pY845 and pY1068 EGFR. The analysis was determined by Western blot a semiquantitative analysis of the expression levels of p‑EGFR (Y845, Y992 and Y1068), β-actin was used as a loading control. Bars show the mean values ± standard deviations of the band density normalized to the total protein. n = 8, *p < 0.05 as compared to control group; #p < 0.05 as compared to liver damage group, respectively using an ANOVA and Tukey’s test

    Article Snippet: The antibodies used in the experiments were as follows: EGFR (1:500, #4267, Cell Signaling Technology, Danvers, MA, USA), phospho-EGFR Y845 (1:500, #2231, Cell Signaling), phospho-EGFR Y992 (1:500, #2235, Cell Signaling), phospho-EGFR Y1068 (1:500, #2236, Cell Signaling), AKT (1:500, #9272, Cell Signaling), phospho-AKT (1:500, #9271, Cell Signaling), STAT5 (1:500, #9363, Cell Signaling), phosphor-STAT5 (1:500, #9351, Cell Signaling), PLC-γ1 (1:500, sc-7290, Santa Cruz Biotechnology, CA, USA), phospho-PLC-γ1 (1:500, sc-136,186, Santa Cruz Biotechnology), β-actin (1:500, sc-47,778 S, anta Cruz Biotechnology), secondary antibody anti-rabbit HRP (1:2000, #7074, Cell signaling, Technology Inc.) and secondary antibody anti-mouse HRP (1:1500, A2304, Sigma-Aldrich).

    Techniques: Expressing, Western Blot