Fig. 5. Monensin causes the formation of large MVBs labeled with the trans-Golgi marker TGN46. (A) Stably transfected K562 cells overexpressing Rab11Q70L were incubated with monensin (1 µM) for 4 hours and with the fluorescent lipid analog N-(lissamine rhodamine B sulfonyl) phosphatidyl ethanolamine (N-Rh-PE) to label exosomes. The lipid was internalized via endocytosis. Cells were mounted on coverslips and immediately analyzed by fluorescence microscopy. Images were obtained as described in Fig. 3. Two sets of images show MVBs that are labeled with the lipid N-Rh-PE (depicted in red) and decorated with GFP-Rab11Q70L (green). (B) Cells overexpressing Rab11Q70L were incubated with monensin (1 µM) for 4 hours. Cells were mounted on coverslips, fixed and subjected to immunofluorescence with a rabbit antibody against TGN46 and a Texas-Red-conjugated goat-anti rabbit secondary antibody. Cells were analyzed by fluorescence microscopy. Two sets of images clearly show that TGN46 (red) colocalizes with the ring-shaped structures decorated by GFP-Rab11Q70L.