Journal: PLoS ONE

Article Title: Identification of the Cancer Cell Proliferation and Survival Functions of proHB-EGF by Using an Anti-HB-EGF Antibody

doi: 10.1371/journal.pone.0054509

Figure Lengend Snippet: A. Binding activity of Y-142 and Y-073 toward sHB-EGF. Anti-HB-EGF antibody was incubated at various concentrations in an sHB-EGF-coated or a BSA-coated plate. Antibody binding was detected with an HRP-labeled anti-mouse IgG antibody. Y-142 binding to BSA, white square; Y-073 binding to BSA, white circle; Y-142 binding to sHB-EGF, black square; Y-073 binding to sHB-EGF, black circle. The data points represent the mean ± standard deviation (SD) of values acquired in duplicate. B. Neutralizing activity of Y-142 and Y-073 against sHB-EGF-induced EGFR phosphorylation. Anti-HB-EGF antibody was incubated at the indicated concentrations in the presence of 10 nM sHB-EGF with NUGC-3 cells for 15 minutes at 37°C. Cell lysates were incubated on an anti-EGFR antibody-coated plate, followed by incubation with HRP-labeled anti-phosphotyrosine antibody. No sHB-EGF, white circle; sHB-EGF and control IgG, white square; sHB-EGF and Y-142, black square; sHB-EGF and Y-073, black circle. The data points represent the mean ± SD of values acquired in triplicate.

Article Snippet: We also used the following reagents: mouse control IgG and horseradish peroxidase-labeled (HRP-labeled) anti-mouse IgG antibody from Jackson ImmunoResearch Laboratories; Alexa488-labeled anti-mouse IgG antibody, HRP-labeled anti-goat IgG antibody, and HRP-labeled anti-rabbit IgG antibody from Invitrogen; anti-amphiregulin (anti-ARG) monoclonal antibody, anti-HB-EGF polyclonal antibody, anti-EGFR polyclonal antibody, and biotinylated anti-EGFR polyclonal antibody from R&D Systems; anti-β-actin antibody from Cell Signaling Technology; erlotinib from Selleck Chemicals; biotinylated anti-phosphotyrosine antibody from Millipore; sulfotagged streptavidin from Meso Scale Discovery; and phorbol 12-myristate 13-acetate (PMA) from Wako.

Techniques: Binding Assay, Activity Assay, Incubation, Labeling, Standard Deviation

Journal: PLoS ONE

Article Title: Identification of the Cancer Cell Proliferation and Survival Functions of proHB-EGF by Using an Anti-HB-EGF Antibody

doi: 10.1371/journal.pone.0054509

Figure Lengend Snippet: The effect of CTF on the observed inhibition of cell proliferation was examined by measuring the amount of CTF. Cell lysates of the PMA-, control IgG, Y-142-, or Y-073-treated NUGC-3 cells were subjected to SDS-PAGE. CTF was detected with anti-CTF polyclonal antibody in western blotting.

Article Snippet: We also used the following reagents: mouse control IgG and horseradish peroxidase-labeled (HRP-labeled) anti-mouse IgG antibody from Jackson ImmunoResearch Laboratories; Alexa488-labeled anti-mouse IgG antibody, HRP-labeled anti-goat IgG antibody, and HRP-labeled anti-rabbit IgG antibody from Invitrogen; anti-amphiregulin (anti-ARG) monoclonal antibody, anti-HB-EGF polyclonal antibody, anti-EGFR polyclonal antibody, and biotinylated anti-EGFR polyclonal antibody from R&D Systems; anti-β-actin antibody from Cell Signaling Technology; erlotinib from Selleck Chemicals; biotinylated anti-phosphotyrosine antibody from Millipore; sulfotagged streptavidin from Meso Scale Discovery; and phorbol 12-myristate 13-acetate (PMA) from Wako.

Techniques: Inhibition, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: Magnetic Nanoparticles as Mediators of Ligand-Free Activation of EGFR Signaling

doi: 10.1371/journal.pone.0068879

Figure Lengend Snippet: Magnetic switches (MS) consisting of SPION covalently coupled to streptavidin further reacted with biotinylated anti-EGFR MAb, in most cases, and biocytin-fluorophore.

Article Snippet: Magnetic switches (MS) capable of targeting EGFR were prepared (except when cells expressed ACP-EGFR, see below) by incubating a 5-fold molar excess of strv-SPIONs, in terms of biotin binding capacity, with biotinylated anti-EGFR monoclonal antibody 528 (Dianova) in 20 mM Na-PO 4 buffer (pH 7.4) at 25°C for 15 min.

Techniques: