Fig. 9. Endocytic uptake of GST-DsRed-RAP in BN16 cells. (A,B) Replicate monolayers of BN16 cells were incubated for 2 hours on ice with phenol-red-free DMEM containing 30 µg/ml of purified GST-DsRed-RAP. Then, the cells were shifted to 37°C and the subcellular localization of GST-DsRed-RAP tested by confocal fluorescence microscopy. At 4°C, all of the GST-DsRed-RAP is bound to the cell surface (A). Internalization of the ligand is seen 20 minutes (B) after shift to 37°C. (C,D) BN16 cells were transiently transfected with an EGFP- MegBP expression construct and incubated in DMEM containing 30 µg/ml of purified GST-DsRed-RAP. After 4 hours, the subcellular localization of EGFP- MegBP and DsRed-GST-RAP was analyzed by confocal fluorescence microscopy. Examples for endocytic uptake of GST-DsRed-RAP in MegBP expressing cells are shown.