Fig. 8. Nocodazole treatment and a 20°C temperature block inhibit the caveolae-mediated endocytosis of CTX to the Golgi but not of AMF to the smooth ER. NIH-3T3 fibroblasts pretreated with 10 µM nocodazole at 37°C (A-K) were pulse labeled with either 5 µg/ml FITC-CTX for 30 minutes at 37°C (A-H) or with 50 µg/ml AMF-FITC for 60 minutes at 37°C (I-K) in the presence of nocodazole. Alternatively, cells were incubated with 50 µg/ml AMF-FITC for 60 minutes at 20°C (L-N). Overlap of endocytosed FITC-CTX (A,D) with anti-GM130 (B), anti-caveolin (E) or anti-TfR (F) labeling and of AMF-FITC (I, L) with anti-AMF-R (J, M) labeling was determined. The merged confocal images (C,G,H,K,N) present the indicated labels in red and green and colocalization in yellow. In the presence of nocodazole FITC-CTX is not delivered to the fragmented Golgi (A-C) but remains associated with caveolin and TfR-positive endosomes (D-H). Neither nocodazole treatment (I-K) nor a 20°C temperature block (L-N) prevent AMF delivery to the smooth ER (arrows). Bar, 8 µm.