FIG. 7.

Inhibition of K1 internalization by LY294002. (A) K1 internalization was measured in the presence or absence of LY294002. Transfected HeLa cells were surface labeled with M1 anti-FLAG, and surface receptors were allowed to internalize for 20 min in the presence of drug or vehicle control (dimethyl sulfoxide). The amount of internalized antibody was quantified by ELISA as described in Materials and Methods. The data are expressed as a ratio of the initial amount of antibody bound to the cell surface at 0 min at 4°C to the amount of antibody bound in the presence of drug after the cells were incubated at 37°C for 20 min. This is depicted as percent internalization on the y axis. The error bars indicate standard deviations. (B) K1 internalization in the presence or absence of LY294002 as analyzed by confocal microscopy. K1-expressing HeLa cells were surface labeled with anti-FLAG-Cy3 and anti-TfR-Alexa 647 for 1 h at 4°C. The surface receptors were allowed to internalize at 37°C for 0, 20, or 60 min in the presence of the drug. The cells were fixed and analyzed by confocal microscopy.