Fig. 2. Hrs and its clathrin-box motif are important for recruitment of clathrin to early endosomes. (A,B) Cells treated with a (A) scrambled control RNA duplex or (B,C) siRNA against Hrs were prepared for immunofluorescence microscopy and labelled with antibodies against clathrin, EEA1 and Hrs. Clathrin was easily detected on EEA1- and Hrs-positive endosomes in control cells (A, inset). In Hrs-depleted cells (arrows), the endosomes were devoid of clathrin (B,C, inset with dashed lines). The siRNA-treated cells were transiently transfected with the siRNA-resistant Hrs constructs indicated, to reintroduce Hrs. Arrowheads point at siRNA-treated cells re-transfected with myc-Hrs_wt (B) or myc-Hrs_CB (C). In siRNA-treated cells transfected with myc-Hrs_wt, endosomal clathrin was detected (B, inset with solid lines). In siRNA-treated cells transfected with myc-Hrs_CB, endosomes were virtually devoid of clathrin (C, inset with solid lines). White in merged images indicates colocalisation of Hrs, EEA1 and clathrin. Bar, 10 µm.