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note
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MDCK cells plated on cover slips were deprived of serum overnight and then microinjected with biotinylated GST fusion peptides containing various SH2 domains. Cells were injected with ∼ 1 fl of 0.5X phosphate-buffered saline containing the indicated peptides or biotinylated GST. After microinjection, cells were incubated at 37°C for 1 hour, and then treated with EGF (100 ng/ml) at 0°C for 15 min (24). The EGF-containing medium was then replaced with warm Dulbecco's modified Eagle's medium, and cells were incubated at 37°C for 30 min. After fixation and permeabilization (24), the cells were incubated first with sheep polyclonal antibodies to EGFRs (Upstate Biotechnology) that had been preabsorbed with GST beads, and then with a mixture of RTC-labeled avidin (to stain microinjected cells) and rhodamine-labeled donkey antibodies to sheep immunoglobulin G (to stain EGFRs) (Jackson Immunology). Cobr photographs were taken by double exposure with two channels (FITC and rhodamine). For quantification of inhibition of EGFR endocytosis, the percentage of injected cells in which EGFR endocytosis was disrupted was determined by multiplying the number of microinjected cells in which EGFR intemalization was blocked by 100 and dividing by the total number of injected cells. For each experiment, 200 to 300 cells were microinjected with a given solution.
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DNA synthesis was assayed after metabolic incorporation of bromodeoxyuridine (BrdU) (Amersham cell proliferation kit). MDCK cells were deprived of serum overnight and then microinjected with antibodies to RAS (2 mg/ml) or biotinylated GRB2 SH2 domain (2 mg/ml). The cover slips were incubated with EGF (50 ng/ml) at 37°C for 6 hours and then with BrdU for 4 hours. BrdU incorporated into DNA was visualized with a rhodamine-labeled secondary antibody to mouse immunoglobulin G. Injected cells were identified by staining with either FITC-labeled antibodies to rat immunoglobulin G or FITC-labeled avidin (Jackson Immunology).
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46
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8944239053
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note
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After serum deprivation overnight and EGF treatment (13), MDCK cells were homogenized. The postnuclear supernatant was centrifuged at 100,000g for 1 hour to yield the soluble and particulate fractions, the latter of which was solubilized with 1% NP-40. The fractions were then subjected to immunoprecipitation with rabbit polyclonal antibodies to GRB2, and the precipitates were washed extensively, resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose filters, and subjected to immunoblot analysis with monoclonal antibodies to GRB2 or to dynamin (Transduction Laboratories). Immune complexes were detected with horseradish peroxidase-conjugated secondary antibodies and chemiluminescence reagents (Amersham). Images were captured on RP film (Kodak) and chemiluminescence was quantified with a Molecular Imager (GS-250; Bio-Rad).
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note
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We thank J. Schlessinger and A. Batzer for GRB2 reagents, D. Bar-Sagi for advice on cell microinjection, and T. Pawson, J. McGlade, M. Tyers, and C. J. Ingles for comments on the manuscript. Supported in part by Ciba-Geigy Canada and grants from the Canadian Cancer Society and Medical Research Council. M.F.M. is a National Cancer Institute of Canada Scientist, and Z.W. is a Charles H. Best Foundation Fellow.
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