FIGURE 5.

Colocalization of EqtII-GFP with BODIPY-TR ceramide. MDCK II cells and NIH 3T3 fibroblasts (FB) transfected with EqtII-GFP or EqtII Y113A-GFP and co-stained with Golgi-specific marker, Bodipy-TR-ceramide (BODIPY). MDCK cells expressing the wild-type EqtII were also treated with 25 μg/ml of D609, while fibroblasts expressing the wild-type EqtII were also treated with BFA at final 50 μg/ml. Scale bar is 20 μm. The colocalization panels show in yellow color the sites where the pixel intensities in both channels are above the calculated threshold values. The framed area is also shown enlarged to reveal details. The BODIPY-GFP scatter-graphs represent the relationship between the intensity of fluorescence in both channels at the same pixel locations in the two channel images. The x axis represents the intensity values 0–255 in the red channel, and the y axis represents the intensity values in the green channel, while the color of the dots represents the frequency of pixels with a given combination of intensities in both channels. The straight white lines represent the automatically calculated threshold values by the method of Costes et al. (25) with the “Colocalisation Threshold” plugin for ImageJ.