Fig. 6. Caveolin-1 overexpression reduces delivery of AMF-FITC to the smooth ER. NIH-3T3 cells were infected with the tTA and caveolin-1 adenoviruses and after 36 hours pulse labeled with AMF-FITC for 60 minutes at 37°C prior to fixation. AMF-FITC was revealed with rabbit anti-FITC followed by Alexa 488 anti-rabbit antibodies (A) and the smooth ER labeled with anti-AMF-R mAb followed by Alexa 647-anti-rat IgM antibodies (B). The caveolin-1-overexpressing cell (arrow) was detected with anti-c-Myc antibodies followed by Texas-Red anti-mouse antibodies (C). The merged confocal image presents AMF-FITC in green, the AMF-R-labeled smooth ER in red and colocalization in yellow (D). Quantification of AMF endocytosis to AMF-R-positive smooth ER tubules (E) shows that caveolin-1-expressing cells exhibit a significant decrease in the caveolae-mediated endocytosis of AMF. Bar, 20 µm.