Fig. 7. AP-2 depletion specifically enhances co-staining of Arf6 cargo with the late endosome and lysosome marker CD63. (A-C) Cells depleted of -adaptin (A), µ2 (B) or clathrin (C) were incubated with antibody against β1 integrin for 4 hours. In C, Alexa-Fluor-546-labelled Tfn was added during the final 30 minutes to identify clathrin-depleted cells. Cells were fixed and subjected to indirect immunofluorescence using anti-CD63 and, for A and B, anti--adaptin. (D) The degree of β1 integrin colocalization with CD63 was quantified using MetaMorph Analysis Software, as detailed in Materials and Methods. The percentage of β1 integrin vesicles colocalizating with CD63 in control cells was determined, then arbitrarily set at 100; the levels in AP-2- and clathrin-depleted cells were expressed relative to this value. All differences were statistically significant (P values ranging from P=0.003 to P=0.009). (E) Enlarged view of the cell labeled with asterisk in the Merge panel of B. CD63, red;, β1 integrin; green. Magnifications of insets are x1.5 (B) and x2 (A,C). Arrows indicate depleted cells.