Fig. 7. Internalized RAP accumulates in the juxtanuclear area. CHO(iLR) cells preincubated with OG-RAP and chased for 30 minutes at 37°C in the presence of 25 µM monensin were fixed and mounted in mowiol. The cells were observed with fluorescence microscopy to visualize RAP that was predominantly located in the juxtanuclear region (A). RAP follows a transferrin-like intracellular pathway. CHO(iLR) cells were simultaneously preincubated with OG-RAP and TMR-Tf and chased for 30 minutes in the presence of 25 µM monensin. Digital images of fixed cells containing RAP (B) and Tf (C) were generated with laser scanning microscopy and the colocalization in the juxtanuclear area was visualized by merging the two images (D). HDLp colocalizes with iLR in the ERC. To determine the localization of iLR after preincubation with OG-HDLp and chase for 30 minutes in the presence of 25 µM monensin, CHO(iLR) cells were fixed and labeled with antibodies against iLR which were visualized with a Cy5-labeled second antibody. OG-HDLp (E) and iLR (F) show significant overlap in the ERC (G). iLR is also abundantly located in the ERC in the absence of ligand or monensin. CHO(iLR) cells were fixed after treatment with incubation medium for 15 minutes at 37°C and iLR was visualized as described above (H). Bars, 10 µm.