Fig. 6.

Effects of multiple KDs on internalization. (A)(Top) Lysates from HeLa KD stable clones (+) for eps15/eps15R, epsin, or eps15/eps15R/epsin (triple KD) or from control clones obtained with mismatched oligos (–) were immunoblotted as indicated. (Middle) EGFR levels were comparable in all clones. (Bottom) The clathrin levels in the clathrin KDs. (B)(Upper) The indicated HeLa KD clones were transfected with a HA-EGFR/Ubmut chimera (see Supporting Materials and Methods) to circumvent the presence of endogenous EGFR. Internalization was assessed with an anti-HA antibody. Data are expressed as the percentage of cells in which internalization was inhibited. (Lower) A HeLa triple KD clone expressing HA-EGFR/Ubmut was transfected with the indicated eps15mut constructs (see Supporting Materials and Methods). Internalization assays and bar graph are as in Upper. Actual images of these experiments are in Fig. 11. (C) HeLa clones harboring the indicated KDs were incubated with 1.5 ng/ml ¹²⁵I-EGF. The rate of internalization is expressed as internalized/surface-bound radioactivity. *, P = 0.0164; **, P = 0.003; linear regression analysis. (D) The indicated HeLa KD clones were treated with filipin (+) or mock treated (–), followed by incubation with 1.5 or 20 ng/ml ¹²⁵I-EGF for 6 min at 37°C. The rate of internalization is expressed as internalized/surface-bound radioactivity.