FIGURE 5.

2ME2 treatment reduces Dab2/clathrin interactions, decreases the movement of endocytic vesicles, and inhibits clathrin exchange. A, lysates from ES-2 cells treated with 2ME2 (4.4 μm, 16 h) or vehicle were immunoprecipitated (ip) with rabbit-α-Dab2 antibody or a specificity control. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with mouse monoclonal α-Dab2, mouse-monoclonal α-clathrin, and rabbit- α-myosin VI. Panel depicts a representative experiment (n = 4). B, ES-2 cells were grown on glass coverslips and treated with 2ME2 (4.4 μm, 16 h) or vehicle. Cells were submitted to live uptake of transferrin (labeled with Alexa-555, 50 μg/ml, 37 °C). After 2 min of continuous uptake, cells were washed with imaging medium, and time lapse series (1 s between acquisitions) of confocal sections of regions adjacent to the cell membrane were acquired (up to 8 min after wash). Bar graph depicts the average ± S.E. of the mean square displacement of 250 vesicles, from 12 different cells per condition, from three independent experiments. C, ES-2 cells were grown on coverslips and transfected with clathrin light chain A fused to GFP (48). 24 h after transfection, cells were treated with 2ME2 (4.4 μm, 16 h) or vehicle. Cells were imaged by confocal microscopy and analyzed by fluorescence recovery after photobleaching (FRAP) (12 cells per condition, three independent experiments), both bleaching and analysis were carried out as in Ref. 49.