Figure 4

The C-terminus of Fas/CD95 mediates trafficking through the Golgi apparatus. (a) Schematic of the Fas/CD95 constructs used (CR, coiled coil region). (b) Fluorescence images of Fas–GFP transiently expressed in HeLa cells. The Golgi was labelled with GRASP65 antibodies. Bar=20 μm. (c) Quantitation of total cellular and Golgi-associated wild-type and tail mutant Fas–GFP measured by fluorescence microscopy (arbitrary units). ^**P<0.01; NS, not significant. (d) Analysis of the proportion of cells with Golgi-associated Fas–GFP (above) and the rate of cell death (below) in response to cycloheximide (CHX) in the absence (right) and presence (left) of Fas ligand, in cells expressing wild-type of tail mutant Fas–GFP. Data show means ± S.D. for three experiments. (e) Analysis of the distribution of tail mutant Fas–GFP in HeLa cells. Note the tubular structures that lie adjacent to, but do not overlap with GRASP65 staining. Further examples of co-localisation with cellular compartments are shown in Supplementary Figure 1. Bar=20 μm (5 μm in zoom). (f) Effect of GRASP65 silencing on Golgi-associated Fas/CD95. Immunoblot showing efficiency of GRASP65 silencing with two oligonucleotides (UTR1, untranslated region 1), and examples images of wild-type and tail mutant Fas–GFP in a GRASP65 silencing background. Bar=10 μm