Fig. 4. Caveolin-1 overexpression negatively regulates CTX internalization to the Golgi apparatus but not to endosomes. NIH-3T3 cells were infected with the tTA and caveolin-1 adenoviruses and after 36 hours pulse labeled with FITC-CTX (A) for 30 minutes at 37°C prior to fixation. Cells were then triple labeled with anti-caveolin antibodies and Alexa 568 anti-rabbit antibodies (B) and anti-GM130 and Alexa 647 anti-mouse antibodies (C). The infected cell is indicated by an arrow, and the merged confocal image (FITC-CTX in green and GM130 in red) shows reduced FITC-CTX internalization to the Golgi in the caveolin-1-infected cell (D). Increased zoom of a caveolin-1-overexpressing cell (E-J) shows internalized FITC-CTX (E) and postfixation caveolin (F) and TfR (G) labeling. Merged confocal images present TfR in green and caveolin in red (H) and FITC-CTX in green and either caveolin (I) or TfR (J) in red. Quantification by mask overlay of FITC-CTX internalization to the GM130-positive Golgi (K) or to TfR-positive endosomes (L) shows that in cells overexpressing caveolin-1, FITC-CTX delivery to the Golgi apparatus is reduced. Bars: A-D in D, 20 µm; E-J in J, 8 µm.