Journal: PLOS One

Article Title: Microfluidic isolation and release of live disseminated breast tumor cells in bone marrow

doi: 10.1371/journal.pone.0319392

Figure Lengend Snippet: (A) Genes were all highly expressed in two cancer cell lines and barely expressed in BM samples. Specifically, there were no detectable levels of EGFR and EpCAM in both BM samples. MDA: MDA-MB-231; MCF7: MCF-7; BM1, BM2: bone marrow cells from healthy donors #1 and #2; BM1+1, +5, +50: 1, 5, 50 MDA-MB-231 cells were spiked into 1 million BM cells from healthy donor #1. (B) EGFR was highly expressed in MDA-MB-231 cells, an observation that aligned with the literature. Nucleated human BM cells were used as control. Streptavidin-PE was used to label biotinylated antibodies.

Article Snippet: Biotinylated antibody against epithelial cell adhesion molecule (anti-EpCAM) (eBioscience, Carlsbad, CA, USA) and biotinylated antibody against epidermal growth factor receptor (anti-EGFR, Research Grade Cetuximab Biosimilar) (R&D Systems, Minneapolis, MN, USA) were used as the tumor cell capture agents immobilized on the surface of the microchannels.

Techniques: Control

Journal: Cellular Signalling

Article Title: Cooperation of distinct Rac-dependent pathways to stabilise E-cadherin adhesion

doi: 10.1016/j.cellsig.2015.04.014

Figure Lengend Snippet: EGFR, but not ErbB3, is required for cadherin-dependent Rac activation. A, Following induction of cell–cell contacts, keratinocyte lysates were immunoprecipitated with anti-E-cadherin antibodies, anti-α3-integrin (positive control) or no antibody (control) and probed with antibodies against proteins shown on the left of panels. B–C, Keratinocytes were treated with EGFR (oligo #1, oligo #2), ErbB3 (E3) or control (scr) siRNA oligos for 48 h. Equal amount of protein was separated on SDS–PAGE and probed with antibodies against proteins shown on the left of each panel. D–I, Keratinocytes were transfected with different siRNA oligos and junctions were initiated for 5 min by the addition of calcium ions and active Rac levels measured. D and G, Proteins were precipitated with GST–PAK-Crib beads (Rac∙GTP) and lysates (Total Rac) were probed with anti-Rac antibodies. The amount of GST fusion protein in each sample was evaluated by Amido Black staining (PAK-Crib). E and H, Depletion of EGFR or ErbB3 are shown. Beta-tubulin is shown as a loading control. F and I, Cell–cell-adhesion-dependent Rac activation was quantified and normalised to Rac·GTP levels at time 0 (no cell–cell contacts) for each siRNA group. Data is representative of 3 independent experiments (thereafter N = 3). *, p < 0.05; **, p < 0.005; n.s., non-significant.

Article Snippet: The following primary antibodies were used against: EGFR (1005; Santa Cruz); ErbB2 (CB11; Serotec); ErbB3 (C17; Santa Cruz); α3-integrin (VM2; gift from F. Watt); E-cadherin (HECD1); α-catenin (VB1); β-catenin (VB2); β1-integrin (gift from F Giancotti); DOCK180 (C19 and H70; Santa Cruz); SOS1 (E1; Santa Cruz); actin (C4; MP Biomedicals); β-tubulin (Tub2.1; Sigma); Rac1 (23A8; Millipore, Upstate); RhoA (26C4; Santa Cruz); and Cdc42 (44; Transduction Laboratories).

Techniques: Activation Assay, Immunoprecipitation, Positive Control, SDS Page, Transfection, Staining

Journal: Cellular Signalling

Article Title: Cooperation of distinct Rac-dependent pathways to stabilise E-cadherin adhesion

doi: 10.1016/j.cellsig.2015.04.014

Figure Lengend Snippet: EGFR depletion perturbs cell–cell contacts. A, Keratinocytes treated with different siRNA oligos were induced to form cell–cell contacts for 30 min, fixed and stained for E-cadherin. Arrows show E-cadherin at junctions; arrowhead points to perturbed junction. B, Western blot shows knockdown of EGFR or ErbB3 (E3). C, Method for quantification of junction disruption. The length of cell–cell contacts (corner to corner) and the length of E-cadherin staining were obtained for each junction and expressed as a ratio (control junctions = 1). D, Disruption of E-cadherin localization by depletion of EGFR. ErbB3 (E3) and non-targeting oligos (con) were used as controls. N = 3 or N = 2 (ErbB3); about 150 junctions quantified in each replicate. Scale bar = 40 μm. *, p < 0.05.

Article Snippet: The following primary antibodies were used against: EGFR (1005; Santa Cruz); ErbB2 (CB11; Serotec); ErbB3 (C17; Santa Cruz); α3-integrin (VM2; gift from F. Watt); E-cadherin (HECD1); α-catenin (VB1); β-catenin (VB2); β1-integrin (gift from F Giancotti); DOCK180 (C19 and H70; Santa Cruz); SOS1 (E1; Santa Cruz); actin (C4; MP Biomedicals); β-tubulin (Tub2.1; Sigma); Rac1 (23A8; Millipore, Upstate); RhoA (26C4; Santa Cruz); and Cdc42 (44; Transduction Laboratories).

Techniques: Staining, Western Blot

Journal: Cellular Signalling

Article Title: Cooperation of distinct Rac-dependent pathways to stabilise E-cadherin adhesion

doi: 10.1016/j.cellsig.2015.04.014

Figure Lengend Snippet: DOCK180 is recruited to E-cadherin complexes. A, Cell–cell contacts were initiated for 5 or 30 min and cells stained for endogenous E-cadherin or DOCK180. Bottom graphs show intensity profile of E-cadherin or DOCK180 by line scan at sites indicated by dashed arrows. B, E-cadherin and DOCK180 staining levels in the junctional area were quantified as a percentage of the total area in the image. C–D, DOCK180 recruitment was assessed to beads coated with anti-E-cadherin or BSA in normal keratinocytes (C) or cells treated with control (con) or EGFR RNAi (D). Beads were added onto cells for 30 min, washed and stained for endogenous DOCK180. E, GST–E-cadherin tail beads were incubated with keratinocyte lysates prepared after a time course of junction induction. Precipitates were probed with anti-DOCK180 antibody (D180); fusion proteins are shown by Comassie Blue (C.B.). F, Following depletion of EGFR, cells were induced to assemble contacts for 15 min and processed as described in E.

Article Snippet: The following primary antibodies were used against: EGFR (1005; Santa Cruz); ErbB2 (CB11; Serotec); ErbB3 (C17; Santa Cruz); α3-integrin (VM2; gift from F. Watt); E-cadherin (HECD1); α-catenin (VB1); β-catenin (VB2); β1-integrin (gift from F Giancotti); DOCK180 (C19 and H70; Santa Cruz); SOS1 (E1; Santa Cruz); actin (C4; MP Biomedicals); β-tubulin (Tub2.1; Sigma); Rac1 (23A8; Millipore, Upstate); RhoA (26C4; Santa Cruz); and Cdc42 (44; Transduction Laboratories).

Techniques: Staining, Incubation

Journal: Cellular Signalling

Article Title: Cooperation of distinct Rac-dependent pathways to stabilise E-cadherin adhesion

doi: 10.1016/j.cellsig.2015.04.014

Figure Lengend Snippet: DOCK180 stabilizes pre-formed junctions. Keratinocytes were treated with siRNA oligos as labelled in the different panels and processed to determine actin recruitment to clustered E-cadherin (A–B) or aggregation assays (C–H). A–B, Following depletion of EGFR or DOCK180, cells were incubated with antibody-coated latex beads for 15 min and stained for F-actin. F-actin clusters are shown by arrows (A) and the proportion of attached beads containing F-actin clusters was quantified and expressed relative to controls (B). C–H, Keratinocytes depleted of DOCK180 (C–E) or SOS1 (F–H) were trypsinised and allowed to aggregate in suspension in the presence of calcium ions for 120 min, followed by disaggregation. C and F, Phase contrast images of initial samples, following aggregation for 120 min and after mechanical stress (disaggr). Black arrows point to aggregates. D and G, Relative sizes of all remaining aggregates were measured and shown in comparison to controls (arbitrarily set as 1). E and H, Confirmation of depletion efficiency following DOCK180 or SOS1 RNAi. Equal amounts of protein were loaded, actin used as a loading control. N = 3. Scale bar = 15 μm (A) or 200 μm (C, F). *, p < 0.03; **, p < 0.0003; ***, p < 0.001; n.s., non-significant.

Article Snippet: The following primary antibodies were used against: EGFR (1005; Santa Cruz); ErbB2 (CB11; Serotec); ErbB3 (C17; Santa Cruz); α3-integrin (VM2; gift from F. Watt); E-cadherin (HECD1); α-catenin (VB1); β-catenin (VB2); β1-integrin (gift from F Giancotti); DOCK180 (C19 and H70; Santa Cruz); SOS1 (E1; Santa Cruz); actin (C4; MP Biomedicals); β-tubulin (Tub2.1; Sigma); Rac1 (23A8; Millipore, Upstate); RhoA (26C4; Santa Cruz); and Cdc42 (44; Transduction Laboratories).

Techniques: Incubation, Staining

Journal: Communications Biology

Article Title: An anion exchange membrane sensor detects EGFR and its activity state in plasma CD63 extracellular vesicles from patients with glioblastoma

doi: 10.1038/s42003-024-06385-1

Figure Lengend Snippet: a Voltage response with anti-CD63 capture and other reporters measured in triplicates. Anti-CD63 reporters and anti-tEGFR reporters give similar responses as they are both present on a vast majority of EVs and because our platform is independent of the affinity of reporter antibody due to the high abundance of reporters. A universal linear calibration between voltage shift and log concentration, corresponding to the linear region of Fig. , is used to estimate the fraction. Each point measured in triplicates. b Four orthogonal approaches using untreated plasma demonstrates that the calibration-free universal correlation for the colocalization fraction \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f=\exp (-\left({V}^{{ref}}-{V}^{{target}}\right)F/2{R}_{B}T)$$\end{document} f = exp ( − V r e f − V t a r g e t F / 2 R B T ) , gives a similar estimate as an independently constructed calibration plot (green bar) and two other orthogonal approaches with ELISA-1 and ELISA-2 (grey bar) defined in our previous work . (F is the Faraday constant, T the temperature, and R B the Boltzmann constant from the Boltzmann theory of ion distribution , V ref and V target are the voltage shift signal with CD63 and EGFR silica reporters.) Also shown, using a PON1 pulldown and quantifying the ubiquitous protein ApoA1 on the pulled-down volume to normalize against the total ApoA1 (blue bar). HDL was used to verify this universality due to its high concentration and ease of validation using orthogonal methods. Same biological replicate was used across all the methods in ( b ). Error bars are one standard deviation.

Article Snippet: Mab806 antibody (ABT-806, Catalog No. TAB-228CL) and Human EGFR (cetuximab) antibody (Catalog No. MAB9577) were purchased from Creative Biolabs, USA, and R&D Systems respectively.

Techniques: Concentration Assay, Clinical Proteomics, Construct, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Standard Deviation

Journal: Biosensors & bioelectronics

Article Title: An aptamer-based magnetic flow cytometer using matched filtering.

doi: 10.1016/j.bios.2020.112362

Figure Lengend Snippet: Figure 5. A. Real-time measurements of Panc-1 model study using E07 aptamer. B. Enumeration plot calculated from MFC measurements, and the inset shows the visualized Panc-1 cells captured by microscope. C. Correlation between mean magnetic intensity (MMI) and mean fluorescence intensity (MFI) with different linkers, anti-EGFR- antibody (shown as Ab) and E07 aptamer, and pancreatic cancer cell lines, Panc-1 and MIA PaCa-2. The error bars represent the counting difference throughout the measurements across 8 addressable sensors.

Article Snippet: A biotinylated anti-EGFR antibody (R&D systems, #FAB9577B-100) was also used for comparison.

Techniques: Microscopy, Fluorescence