Fig. 2. Effect of GAK depletion on receptor-mediated endocytosis and MPR trafficking. (A) Uptake of transferrin is inhibited in GAK-depleted cells. Alexa Fluor 546-transferrin (10 µg/ml) was incubated with the cells for the times indicated. (a-d) Mock-depleted and (e-h) GAK-depleted cells. (B) Internalization of ¹²⁵I-transferrin was measured as a function of time in mock-depleted (open circles) and GAK-depleted cells (solid circles). The total transferrin is the amount bound plus the amount endocytosed and later exocytosed. The initial surface values for both the control and GAK-depleted cells were about 3500 c.p.m. (C) Efflux of ¹²⁵I-transferrin was measured as a function of time in mock-depleted (open circles) and GAK-depleted cells (solid circles). Uptake and efflux of transferrin was performed as described in the Materials and Methods. (D) Uptake of EGF is inhibited in GAK-depleted cells. Cells were incubated with Alexa Fluor 647 EGF (4 µM) for 30 minutes in mock-depleted (a) and GAK-depleted (b) cells. (E) Localization of MPR is altered after GAK-depletion. MPR was immunostained in mock-depleted (a) and GAK-depleted (b) cells.