Fig. 5. CTX endocytosis in clathrin-hub-expressing cells. NIH-3T3 cells infected for 36 hours with the tTA and clathrin hub adenoviruses were incubated at 37°C with FITC-CTX for 5 (A-D) or 30 minutes (E-H). The distribution of FITC-CTX was visualized directly (A,E) and post-fixation labeling with anti-caveolin (B,F) and anti-GM130 (C,G) antibodies revealed with the appropriate Alexa-568- and Alexa-647-conjugated secondary antibodies, respectively. Merged confocal images (D,H) present FITC-CTX in green, caveolin in red and GM130 in blue. After five minutes of endocytosis, CTX is primarily associated with caveolae and with time accumulates in the perinuclear region, where, after 30 minutes, it colocalizes extensively with the Golgi apparatus. Bar, 8 µm.