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Fig. 3. Effect of cell-permeable Antennapedia fusion peptides that mimic the Fer-p120ctn binding site on in vitro binding between Fer and p120ctn, and on the composition of the cadherin complex of proteins in E8 chick retina cells. (A) Biotin-labeled CBP-p120ctn was immobilized on streptavidin wells and incubated with 3 µg GST-Fer per well in the presence of increasing concentrations of competing peptides. Bound Fer was estimated using anti-GST antibody. Abbreviations: FerP and FerCo, Antennapedia cell-permeable peptides covalently attached to the Fer-7 or the reverse Fer-7 sequence, respectively; p120P and p120Co, Antennapedia peptides covalently attached to the P10 or the reverse P10 sequence. (B) Intact E8 retina cells were incubated in the presence of the indicated peptide for 45 minutes, 60 minutes or 120 minutes at room temperature. The cells were homogenized in buffer containing 1% NP-40 and immunoprecipitated with NCD-2. The resulting immuno-complexes were analysed by western blot with the indicated antibody. (C) Intact E8 retina cells were incubated in the presence of the indicated peptide for 60 minutes at room temperature, lysed in RIPA buffer, immunoprecipitated with anti-ß-catenin antibody and analysed by western blot using anti-phosphotyrosine antibody. The blots were then stripped and blotted with anti-ß-catenin antibody. The relative intensity of the ß-catenin western blot bands was measured by densitometry and is presented as the ratio between phosphotyrosine ß-catenin and total ß-catenin. (D) Retina cells were incubated with the indicated peptides and labeled with a cell-impermeable biotinylation reagent for the indicated time. The cells were lysed as above, cell-surface N-cadherin pulled down with avidin-coated beads, fractionated by SDS-PAGE and visualized by western blot with anti-N-cadherin antibody. An aliquot of the total lysate was also analyzed by western blot, for comparison.
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