mouse anti phosphorylated egfr  (Cell Signaling Technology Inc)

Journal: Molecules (Basel, Switzerland)

Article Title: Deacetylated Sialic Acid Sensitizes Lung and Colon Cancers to Novel Cucurbitacin-Inspired Estrone Epidermal Growth Factor Receptor (EGFR) Inhibitor Analogs.

doi: 10.3390/molecules28176257

Figure Lengend Snippet: Figure 1. (A) Non-canonical forms of Sia (Deacetyl Sia, Acetyl Sia, KDN = Deaminated Sia and Neu5Gc = N-Glycolyl Neuraminic acid) implicated in several biological processes. (B) CASD1 enzyme mediates 9-O and 7,9-O acetylation of Sia (Neu 5Ac). CASD1 adds acetyl functional groups, via an Acetyl CoA donor, to the seventh carbon of Sia, from which it migrates to the ninth carbon (Neu 5,9Ac2) under physiological conditions. (C) Absence of CASD1 abrogates the addition of acetyl groups to Sia residues on the EGFR glycan chain, resulting in the overexpression of activated (phosphorylated) EGFR, increased levels of activated downstream proteins such as ERK and mTOR and increased proliferation of cells. (D) Treatment of CASD1-deficient/deacetylated Sia-expressing cells with CIEAs (i.e., EGFR inhibitor) inhibits EGFR activation, decreases levels of phosphorylated (activated) downstream proteins (i.e., mTOR and ERK), decreases cell proliferation and induces apoptosis as well as G2/M cell cycle arrest/switch.

Article Snippet: The slides were blocked in a buffer containing 0.01% goat serum, 0.01% saponin, and 0.05% glycine in PBS for 1 h and incubated with human phosphorylated EGFR monoclonal IgG1 mouse antibody (Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 40C.

Techniques: Functional Assay, Glycoproteomics, Over Expression, Expressing, Activation Assay

Journal: Molecules (Basel, Switzerland)

Article Title: Deacetylated Sialic Acid Sensitizes Lung and Colon Cancers to Novel Cucurbitacin-Inspired Estrone Epidermal Growth Factor Receptor (EGFR) Inhibitor Analogs.

doi: 10.3390/molecules28176257

Figure Lengend Snippet: Figure 6. Effects of Sia deacetylation on Phosphorylated EGFR (pEGFR) expression. Cells were fixed, permeabilized, treated with pEGFR-specific primary antibody and Alexa-Fluor 488 conjugated secondary antibody. DAPI was used for counter staining. (A) Immunofluorescent localization of pEGFR in A549 wild type 6A(A1–A3) and CASD1 Knockout 6A(A4–A6) cell lines. A1&A4: DAPI stained nuclei, A2&A5: pEGFR fluorescence and A3&A6: Merged DAPI stained nuclei and fluorescent pEGFR (B) pEGFR fluorescence intensity for A549 cell lines. (C) Immunofluorescent localization of pEGFR in HCT 116 wild type 6C(C1–C3) and CASD1 Knockout 6C(C4–C6) cell lines. A1&A4: DAPI stained nuclei, A2&A5: pEGFR fluorescence and A3&A6: Merged DAPI stained nuclei and fluorescent pEGFR (D) pEGFR fluorescence intensity plot for A549 cell lines. One-way ANOVA with Tukey’s post-test was used for multiple plot comparisons, ** p < 0.01 and *** p ≤0.001.

Article Snippet: The slides were blocked in a buffer containing 0.01% goat serum, 0.01% saponin, and 0.05% glycine in PBS for 1 h and incubated with human phosphorylated EGFR monoclonal IgG1 mouse antibody (Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 40C.

Techniques: Expressing, Staining, Knock-Out

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Biological and clinical impact of membrane EGFR expression in a subgroup of OC patients from the phase IV ovarian cancer MITO-16A/MANGO-OV2A trial.

doi: 10.1186/s13046-023-02651-y

Figure Lengend Snippet: Fig. 2 Kaplan-Meyer analysis evaluating the progression free survival (PFS) and the overall survival (OS) of patients from the MITO16a-MaNGO-OV2i stratified according to EGFR membrane expression by immunohistochemical staining

Article Snippet: The primary antibodies used were: mouse anti-phosphorylated EGFR (Tyrosine 1068) (1H12, Cell Signaling.

Techniques: Membrane, Expressing, Immunohistochemical staining, Staining

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Biological and clinical impact of membrane EGFR expression in a subgroup of OC patients from the phase IV ovarian cancer MITO-16A/MANGO-OV2A trial.

doi: 10.1186/s13046-023-02651-y

Figure Lengend Snippet: Fig. 1 Representative images of OC sections stained with the anti-EGFR antibody. C, cytoplasmic staining; NEG, no staining. Percentage of the clear cell membrane staining is reported below pictures. A Representative images of an entire section within the TMA is reported. Black empty box, area shown in panel B. Bar, 200 μm. B Higher magnification of each section in panel A

Article Snippet: The primary antibodies used were: mouse anti-phosphorylated EGFR (Tyrosine 1068) (1H12, Cell Signaling.

Techniques: Staining, Membrane

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Biological and clinical impact of membrane EGFR expression in a subgroup of OC patients from the phase IV ovarian cancer MITO-16A/MANGO-OV2A trial.

doi: 10.1186/s13046-023-02651-y

Figure Lengend Snippet: Fig. 3 A Graphical representation of the mean GSVA scores for the EGFR-related gene sets (see Table 3 for GSVA scores and GSEA). The red line highlights the 0 score. Different dots’ colors represent different gene sets, as reported. For the corresponding GSEA nomenclature refers to Table 4. B Graphical representation of the overlap among the gene sets significantly enriched in MM staining subgroup defined in the MITO16a-MaNGO-OV2 trial. The three gene sets (a), (d) and (e) were selected for further analysis since they include all the EGFR-related genes. Each black line represents a gene; for each gene set the number of genes is reported at the bottom. The names of the gene sets are reported below the scheme

Article Snippet: The primary antibodies used were: mouse anti-phosphorylated EGFR (Tyrosine 1068) (1H12, Cell Signaling.

Techniques: Staining

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Biological and clinical impact of membrane EGFR expression in a subgroup of OC patients from the phase IV ovarian cancer MITO-16A/MANGO-OV2A trial.

doi: 10.1186/s13046-023-02651-y

Figure Lengend Snippet: Fig. 4 Graphical representation of the genes/functions predicted by IPA performed using the differential gene expression derived by comparing MM vs M and MO groups of patients. IPA was run using the log fold changes of the genes up- or down-modulated in MM vs the other two subgroups and included in the EGFR-related gene sets reported in Table 4. See Methods for details of the analysis. The name of each gene set is reported above each scheme. Panel in the right side, prediction legends

Article Snippet: The primary antibodies used were: mouse anti-phosphorylated EGFR (Tyrosine 1068) (1H12, Cell Signaling.

Techniques: Gene Expression, Derivative Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Biological and clinical impact of membrane EGFR expression in a subgroup of OC patients from the phase IV ovarian cancer MITO-16A/MANGO-OV2A trial.

doi: 10.1186/s13046-023-02651-y

Figure Lengend Snippet: Fig. 6 A Western blotting for EGFR and AXL expressions in lysates from OC cell lines representative of HGSOCs or non-HGSOCs cell lines [34]. SKOV3 and OVCAR5 cells co-expressed both EGFR and AXL. B Confocal immunofluorescence showing EGFR and Axl expressions; only SKOV3 showed both EGFR (green) and AXL (red) expressions on the cell membrane with several regions of co-localization (white box indicates one of those regions). In OVCAR5 cells only EGFR is clearly expressed on the membrane. SKOV3 was chosen for further analysis. Upper, merge staining; lower. single staining, Nuclei were stained with DAPI. C upper. Western blotting of lysated from SKOV3 cells stimulated with GAS6 or EGF alone or in combination; lower, densitometric analysis for phoshorylated (P-EGFR and P–AXL) or total RTKs (EGFR and AXL). As expected, the total amount of EGFR decreased upon ligand stimulation [35]. No changes in the amount of AXL were detected upon GAS6 stimulation. D Erlotinib susceptibility of AXL silenced SKOV3 cells. Upper, western blotting showing the amount of silenced AXL in SKOV3 lysates upon transfection with two different siRNAs (#1 and #2). Lower, viability of SKOV3 cells treated with control siRNA (siCO) or with specific Axl siRNAs (siAxl#1 and #2) and then treated with erlotinib at different concentration. Statistical evaluation by ANOVA, p ≤ 0.001. Refer to Methods section for detailed procedure. The table below reports the IC50 values of siRNA transfected cells. The experiments were performed at least three times

Article Snippet: The primary antibodies used were: mouse anti-phosphorylated EGFR (Tyrosine 1068) (1H12, Cell Signaling.

Techniques: Western Blot, Immunofluorescence, Membrane, Staining, Transfection, Control, Concentration Assay

Journal: Nature communications

Article Title: Counting growth factors in single cells with infrared quantum dots to measure discrete stimulation distributions.

doi: 10.1038/s41467-019-08754-5

Figure Lengend Snippet: Fig. 4 Counting and simulating growth factor binding. a Representative z-projections of maximum intensity (left) and three-dimensional (3D) images (right) of cells treated with indicated quantum dot-epidermal growth factor (QD-EGF) concentration for 5 min on ice. b Number of QD-EGF bound per cell at indicated QD-EGF concentrations, showing two independent replicates with N ≥17 cells for each condition. c Distributions show the number of EGF per cell measured experimentally at the indicated QD-EGF concentration. Maximum likelihood estimation regressions of gamma distributions are shown as red lines and simulation results are shown as blue lines. For regression, p = 0.79, 0.88, and 0.85 for 0.1, 1, and 10 nM QD-EGF concentrations, respectively. For simulation, p = 0.24, 0.49, and 0.67 for 0.1, 1, and 10 nM QD-EGF concentrations, respectively. All p values were calculated using χ2 tests. d EGF per cell is shown as experimental results (gray) and simulation results (blue) across different concentrations. Simulation results in d were obtained by sampling cells from the EGFR number gamma distribution (see Methods). e Representative z-projections of maximum intensity of breast cancer cell lines MCF-7, MDA- MB-231, and MDA-MB-468 in order of increasing EGFR expression. Cells were treated with 1 nM QD-EGF for 5 min on ice. Yellow arrow indicates a single QD-EGF bound to an MCF-7 cell. f Number of QD-EGF bound per cell for conditions in e with N ≥40 cells for each condition. QDs are shown in red and nuclei are blue. In a, e, QDs are shown in red and nuclei are blue; scale bars, 10 µm. In b, d, f, the box indicates 25/75th percentile; red lines are means; whiskers are s.d.

Article Snippet: Mouse monoclonal IgG antibody against phosphorylated EGFR was purchased from R&D Systems.

Techniques: Binding Assay, Concentration Assay, Sampling, Expressing

Journal: Nature communications

Article Title: Counting growth factors in single cells with infrared quantum dots to measure discrete stimulation distributions.

doi: 10.1038/s41467-019-08754-5

Figure Lengend Snippet: Fig. 5 Single-cell epidermal growth factor (EGF) binding correlates with single-cell receptor translocation and drug response. a Representative three- dimensional (3D) images of MDA-MB-231 cells after stimulation with quantum dot-EGF (QD-EGF) in the absence or presence of EGFR inhibitor gefitinib. Times after the start of a stimulation pulse are indicated. QDs are shown in red, nuclei are blue, and Alexa Fluor 488-conjugated fibronectin micropatterns are green. b Two-dimensional (2D) z-projections on xy fibronectin micropattern planes and c one-dimensional (1D) projections on x-axes indicate the localization of single EGF averaged across cells. d Representative image of cell membrane measured through fluorescence imaging of fluorescently labeled receptors (top) and membrane reconstruction using alpha shapes (bottom). e Correlation between EGF number and fraction of EGF internalized in individual cells at 10 and 30 min after the start of a QD-EGF stimulation pulse. f Western blots and g relative pEGFR abundance in MDA-MB-231 whole-cell lysates immediately after stimulation with QD-EGF in the presence of indicated gefitinib concentrations. Uncropped western blots with molecular weight markers are shown in Supplementary Figure 16. h Fraction of EGF internalized in single cells at different gefitinib concentrations, 30 min after the start of a QD-EGF pulse. The box indicates 25/75th percentile; red lines are means; whiskers are s.d. i Coefficient of variation (CV) of the fraction of EGF internalized in h. j Number of EGF bound impacts the fraction of EGF internalized 10 min after the start of a QD-EGF pulse in the presence of gefitinib at 0, 51, and 5,100 nM concentration. The gray line shown in 51 nM (middle) and 5100 nM (right) gefitinib plots is the linear fit for 0 nM gefitinib condition (left). Data fits are shown in Supplementary Figure 14. N = 20 and 12 cells for 10 and 30 min after QD-EGF stimulation onset without gefitinib, respectively; N = 12, 10, 20, 23, 12, and 14 cells for 30 min after QD-EGF stimulation onset in the presence of gefitinib at 0, 0.51, 5.1, 51, 510, and 5100 nM concentrations, respectively. All stimulation pulses used 1 nM EGF-QD for 5 min. All scale bars indicate 10 µm

Article Snippet: Mouse monoclonal IgG antibody against phosphorylated EGFR was purchased from R&D Systems.

Techniques: Binding Assay, Translocation Assay, Membrane, Imaging, Labeling, Western Blot, Molecular Weight, Concentration Assay