Fig. 5. Vesicles containing internalized anti-PRL recycle in a stimulus-dependent manner. To measure recycling, cells that had internalized anti-PRL in response to a first stimulation were sequentially incubated with Cy2-labeled secondary antibodies in standard external solution for 30 minutes and then Cy3-labeled secondary antibodies in high [K⁺] external solution for 5 minutes. Anti-PRL that recycled to the cell surface in a basal manner was labeled with Cy2 (green). Granules that recycled back to the cell surface in a stimulus-dependent manner were labeled with Cy3 only (red). (A) Basal recycling (green) measured from t=50 to 80 minutes; stimulus-dependent recycling measured at t=80. (B) Basal recycling (green) measured from t=80 to 110 minutes; stimulus-dependent recycling measured at t=110. (See Materials and Methods for details of the assay and Table 1 for quantitation.) DAPI (blue) was included in the mounting media. The images shown are projection images from the entire z-series. Bar, 10 µm.