Figure 3.

AAK1L CBD2 overexpression impairs transferrin internalization and disrupts perinuclear clathrin localization. Tetracycline transactivator (tTA) HeLa cells were infected with control adenovirus encoding the (tTA, control) or AAK1L CBD2 for 24 h. Cells were incubated at 4°C for 1 h with biotinylated transferrin (BXX-Tfn) to allow binding to the transferrin receptor, washed, and transferred to 37°C to promote a single round of transferrin internalization (see Materials and Methods). Error bars, the SD of three independent experiments. (B) tTA HeLa cells were infected with the indicated control and AAK1-encoded adenoviruses. Twenty-four hours after infection, cells were fixed, permeabilized, and processed for clathrin immunolocalization. Clathrin was detected by epifluorescence using the mAb TD.1 and an appropriate fluorochrome-tagged secondary antibody. Cells were visualized using a Zeiss Axioscop 2 with attached Axiocam MRm.