Fig. 3. Depletion of -adaptin and µ2 subunits of AP-2 blocks endocytosis of TfnR. (A) HeLa cells were transfected with the indicated siRNA oligonucleotides. Whole-cell lysates were prepared and immunoblotted with antibodies against -adaptin, µ2-subunit or TfnR. (B) Live control cells were incubated with Alexa-Fluor-546-conjugated Tfn, then subjected to immunofluorescence using anti--adaptin antibody. (C) Live -adaptin-depleted cells were incubated with Alexa-Fluor-488–Tfn, then stained with antibodies against -adaptin and TfnR. (D) µ2-depleted cells were incubated with Alexa-Fluor-546-labeled Tfn, fixed, and then subjected to indirect immunofluorescence using anti--adaptin antibody. In C and D, the depleted cells are in the upper left parts of the panels.