Journal:

Article Title: Grb2 Regulates Internalization of EGF Receptors through Clathrin-coated Pits

doi: 10.1091/mbc.E02-08-0532

Figure Lengend Snippet: Schematic representation of EGF receptor mutants. (A) Wild-type EGFR (WT) is drawn with the extracellular, transmembrane (TM) and intracellular domain (kinase and C terminus). Main tyrosine phosphorylation sites (residues 992, 1068, 1086, 1148, and 1173) and a Cbl binding site (Tyr1045) are indicated in WT receptor. Mutants have C-terminal truncation of 79 (C′1107), 96 (C′1090), 114 (C′1072), 123 (C′1063), and 164 amino acid residues (C′1022). Tyrosine substitutions by phenylalanines (F) are indicated. (B) To confirm the correct size of truncated EGFR mutants, PAE cells expressing WT, C′1107, C′1090, C′1072, C′1063, or C′1022 were lysed, and the EGFR was detected in lysates by immunoblotting with antibody 2913.

Article Snippet: Internalization of 125 I-EGF and 125 I-transferrin Mouse receptor-grade EGF was obtained from Collaborative Research Inc. (Bedford, MA) and iodinated using a modified chloramine T method as described previously ( Sorkin et al. , 1991 ).

Techniques: Binding Assay, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Quantitation of the Effect of ErbB2 on Epidermal Growth Factor Receptor Binding and Dimerization *

doi: 10.1074/jbc.M112.373647

Figure Lengend Snippet: Binding of 125I-EGF to cells expressing EGF receptors and ErbB2. A, 125I-EGF binding to cells expressing ∼300,000 EGF receptors and the indicated number of ErbB2 molecules. The level of ErbB2 was measured by the binding of 125I-trastuzumab. B, model for the binding of EGF to EGFR homodimers and EGFR/ErbB2 heterodimers. Open circles represent EGF receptor molecules. Hatched circles represent ErbB2 molecules. E is a bound EGF molecule. The term that refers to the equilibrium association constant for each interaction is indicated over the arrows. Fitted values for the equilibrium association constants are shown. Values in red indicate the values that were fitted to Equation 1 based on the binding data in panel A. Values in black were set as constants during the fitting and are based on previous studies of EGF binding to only the EGF receptor. Values in gray were calculated based on the principle of microscopic equilibrium.

Article Snippet: 125 I-EGF and 125 I-Trastuzumab Synthesis and Binding Analyses Murine EGF (Biomedical Technologies, Inc.) and trastuzumab (Barnes Hospital pharmacy) were radiolabeled with 125 I using the ICl method ( 24 ).

Techniques: Binding Assay, Expressing

Journal:

Article Title: Sprouty2 attenuates epidermal growth factor receptor ubiquitylation and endocytosis, and consequently enhances Ras/ERK signalling

doi: 10.1093/emboj/cdf493

Figure Lengend Snippet: Fig. 3. hSpry2 abrogates c-Cbl-dependent ubiquitylation of EGFRs. (A) COS-1 cells were co-transfected with 1.0 µg HA-tagged ubiquitin and 3.0 µg each of either vector control, c-Cbl, c-Cbl-C381A or the different FLAG-tagged Spry forms per 100 mm culture dish. Forty-eight hours later, cell monolayers were treated with 100 ng/ml EGF at 37°C for 10 min. Total cell lysates (TCL) were subjected to immunoprecipitation (IP) using anti-EGFR–agarose-conjugated beads, immunoblotted (IB) with anti-HA to distinguish ubiquitin-conjugated EGFR, and anti-EGFR to assess the amounts of immunoprecipitated EGFR. TCL samples were analysed with anti-HA or anti-FLAG to show relative expression levels of the various constructs. The bracket indicates the position of high molecular weight species of ubiquitin-positive EGFRs. (B) Immunoprecipitated EGFR proteins were subjected to an in vitro ubiquitylation assay in the presence of purified UbcH7 (or no added UbcH7 as control without E2), eluted c-Cbl protein alone (or no added c-Cbl as control without E3), recombinant c-Cbl with either GST–hSpry2, GST–mSpry4 or GST–hSpry2ΔN11 fusion proteins, or c-Cbl-C381A fusion protein alone, plus the essential components in the ubiquitylation system. The reaction products were analysed following western blotting protocol with anti-ubiquitin. The bracket highlights the position of high molecular species of ubiquitin-positive EGFRs. (C) Competitive binding between hSpry2 and UbcH7 for the RING finger domain of c-Cbl. COS-1 cells (100 mm dishes) were co-transfected with 3.0 µg each of FLAG-tagged UbcH7, HA–c-Cbl or HA–c-Cbl-ΔRF mutant, and varying amounts of FLAG–hSpry2 or the c-Cbl non-binding truncation mutant FLAG–hSpry2ΔN11, as indicated. Serum-deprived cells were stimulated with 100 ng/ml EGF at 37°C for 10 min. TCL were subjected to precipitation using anti-HA and immunoblotted (IB) with anti-FLAG to detect associated hSpry2 or UbcH7, and anti-HA to show the relative amounts of immunoprecipitated c-Cbl. A TCL blot was probed with anti-FLAG to demonstrate quantitatively the expression levels of exogenous hSpry2 and UbcH7 proteins. (D) COS-1 cells (100 mm dishes) were untransfected (control), or co-transfected with 3.0 µg each of FLAG-tagged UbcH7 and either HA–hSpry2, HA–human Ariadne-2 (hARI-2), HA–Drosophila Ariadne-1 (dAri-1), HA–c-Cbl or HA–c-CblΔRF constructs. Immunocomplexes of FLAG–UbcH7 or ‘pull-downs’ using GST–hSpry2 were detected using anti-HA. Total cell lysates were analysed for equivalent levels of protein expression and normalized sample loading using antibodies as indicated. An immunoblot (with TCL loaded alongside) was probed with anti-HA to detect hSpry2-binding proteins, and with anti-FLAG to verify equal amounts of immunoprecipitated UbcH7.

Article Snippet: 125 I-labelled human recombinant EGF was from Amersham Pharmacia Biotech (Buckinghamshire, UK).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, Construct, Molecular Weight, In Vitro, Ubiquitin Assay, Purification, Recombinant, Western Blot, Binding Assay, Mutagenesis