Journal: bioRxiv

Article Title: Distinct interactions stabilize EGFR dimers and higher-order oligomers in cell membranes

doi: 10.1101/2023.04.10.536273

Figure Lengend Snippet: a Schematic diagram of epidermal growth factor receptor (EGFR) defining the major domains: extracellular region (ECR), intracellular region (ICR), transmembrane domain (TM), intracellular juxtamembrane domain (JM), tyrosine kinase domain (TKD), and the C-terminal tail (C-Tail). b Single-particle tracking (SPT) was performed using streptavidin-quantum dots (QD) conjugated to biotinylated HA antibody Fabs (QD-HA) or to biotinylated EGF (QD-EGF) as detailed in the Methods. Unless otherwise specified, cells were stimulated and tracked using QD-EGF at 200 pM. Time series were acquired at 46 Hz and individual receptors were tracked over time. c Representative 2D trajectories are shown for individual receptors diffusing on the cell membrane, from which diffusion coefficients ( D ) were calculated for each receptor and averaged over the cell (see Methods). d HA-tagged wild type EGFR expressed in CHO cells was tracked without ligand stimulation using QD-HA (gray open diamonds) or following stimulation with 200 pM QD-EGF (gray filled diamonds). The mean measured D value for each cell (n ≥ ∼50 tracks per cell) is plotted. Mean (± SD) across all cells are indicated (see Supplementary Table 1), and distributions are compared using Welch’s t-test ( P = 1.5 × 10 -9 ). All source data are provided as a Source data file.

Article Snippet: For signaling studies, primary antibodies were all used at a 1:1,000 dilution as follows: for phosphorylated EGFR (pY1173: CST#4407), total EGFR (R&D AF231) and ERK1/2 (pT202/pY204: CST #9106; total ERK: CST#4696).

Techniques: Single-particle Tracking, Diffusion-based Assay

Journal: bioRxiv

Article Title: Distinct interactions stabilize EGFR dimers and higher-order oligomers in cell membranes

doi: 10.1101/2023.04.10.536273

Figure Lengend Snippet: a Schematic of mutations introduced to disrupt the Sm-x-x-x-Sm dimerization motifs in the EGFR TM domain. There are three Sm-x-x-x-Sm motifs, where Sm is any small amino acid, in EGFR (T-g-m-v-G; G-m-v-g-A; and A-l-g-i-G), with the small residues colored red. All Sm residues were mutated to valine in TM3X. b SPT data for wild type EGFR (gray) and a variant harboring TM mutations that block TM domain dimerization (TM3X: blue). HA-tagged receptors were labeled and tracked with QD-HA in the absence of ligand (open diamonds), and using 200 pM QD-EGF to investigate the effect of ligand binding (filled diamonds). EGF induces a very similar and significant slow-down for both wild type EGFR ( n = 40 without ligand, 42 with ligand; P = 6.7 × 10 -6 : see Supplementary Table 3) and the TM3X variant ( n = 45 without ligand, 46 with ligand; P = 1.9 × 10 -7 ). Unpaired two-sided Welch’s t-tests were used to calculate P values (** P < 1×10 -3 ; *** P < 1×10 -6 ). c pEGFR immunoblots of wild type and TM3X EGFR activated with 16 nM EGF for 5 min and probed with anti-pY1173 (upper), anti-EGFR (middle) and anti-GRB2 (lower) as loading control. Representative of three biological repeats. All source data and uncropped gels (including total EGFR and GRB2 and tubulin loading controls) are provided in a Source data file.

Article Snippet: For signaling studies, primary antibodies were all used at a 1:1,000 dilution as follows: for phosphorylated EGFR (pY1173: CST#4407), total EGFR (R&D AF231) and ERK1/2 (pT202/pY204: CST #9106; total ERK: CST#4696).

Techniques: Variant Assay, Blocking Assay, Labeling, Ligand Binding Assay, Western Blot

Journal: bioRxiv

Article Title: Distinct interactions stabilize EGFR dimers and higher-order oligomers in cell membranes

doi: 10.1101/2023.04.10.536273

Figure Lengend Snippet: a Structural view of asymmetric dimer between two EGFR TKDs from PDB ID: 3GOP 9 . The C-lobe of the activator kinase (yellow) interacts with the N-lobe of the receiver kinase, leading to its allosteric activation . The asymmetric dimer interface between receiver and activator is augmented by the JM ‘latch’ 6,9 , with key interacting residues colored cyan (see left insert). The JM4X mutation includes replacing L664, V665, L668, and S671 all with alanine to disrupt JM interactions. Mutations to disrupt the asymmetric dimer interface are I682Q in the receiver N-lobe (purple) and V924R in the activator C- lobe (dark pink). b SPT analysis of wild type EGFR (gray) and the JM4X variant (cyan). QD-EGF (200 pM) induces a significant slow-down for wild type ( P = 9.3 × 10 -4 ; n = 32 without ligand, 34 with ligand), but this is lost in the JM4X variant ( P = 0.91; n = 24 without ligand, 30 with ligand). Unpaired two-sided Welch’s t-tests were used to calculate P values (** P < 1×10 -3 ; n.s. indicates no significant difference), listed in Supplementary Table 3. c pEGFR and pERK immunoblots of wild type and JM4X EGFR activated with 16 nM EGF for 5 min and probed with anti-pY1173, anti-EGFR, anti-pERK, anti-ERK, and anti- GRB2 (lower) as loading control. Source data and uncropped gels are provided in the Source data file. Representative of three biological repeats. The JM4X still shows a small degree of signaling at pEGFR and pERK levels, but it is greatly reduced compared with wild type. d SPT analysis of wild type EGFR (gray), the I682Q N-lobe-mutated variant (purple) and the V924R C-lobe mutated variant (dark pink). QD-EGF (200 pM) induces a significant slow-down for wild type in these experiments ( P = 1.9 × 10 -9 ; n = 126 without ligand, 118 with ligand), but this is lost in both the I682Q variant ( P = 0.13; n = 116 without ligand, 116 with ligand) and the V924R variant ( P = 0.36; n = 108 without ligand, 124 with ligand). Unpaired two-sided Welch’s t-tests were used to calculate P values (*** P < 1×10 -6 ; n.s. indicates no significant difference), listed in Supplementary Table 1. All source data are provided as a Source data file.

Article Snippet: For signaling studies, primary antibodies were all used at a 1:1,000 dilution as follows: for phosphorylated EGFR (pY1173: CST#4407), total EGFR (R&D AF231) and ERK1/2 (pT202/pY204: CST #9106; total ERK: CST#4696).

Techniques: Activation Assay, Mutagenesis, Variant Assay, Western Blot

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

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

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

Journal: Metabolites

Article Title: Glucolipotoxic Stress-Induced Mig6 Desensitizes EGFR Signaling and Promotes Pancreatic Beta Cell Death

doi: 10.3390/metabo13050627

Figure Lengend Snippet: List of antibodies used for immunoblotting.

Article Snippet: Anti-phospho-EGFR (Tyr1068) , Cell Signaling, #3777 , 1:1250.

Techniques: Western Blot