Fig. 8. Endocytosis of Rh-EGF is inhibited in cells expressing GFP-Rab21 T33N. HeLa cells transfected with GFP-Rab21 plasmids using calcium phosphate were incubated for 30 minutes at 37°C with 150 ng/ml Rh-EGF, washed on ice, fixed and analysed by confocal fluorescence microscopy. There was no visible difference in the levels or distribution pattern of internalised Rh-EGF (red in merge) in nontransfected cells and those expressing GFP-Rab21 wt (A-C) or GFP-Rab21 Q78L (D-F). Levels of internalised EGF were significantly lower in cells expressing GFP-Rab21 T33N and the label was confined to relatively small vesicles (G-I). Arrows in A-F point to Rab21 wt and Rab21Q78L colocalisation with internalised Rh-EGF. Asterisks represent transfected cells. Bars, 10 µm. Video microscopy of HeLa cells expressing GFP-Rab21 wt, GFP-Rab21 Q78L and GFP-Rab21 T33N following 15 minutes internalisation of Rh-EGF can be seen as QuickTime Movies 4-6 in supplementary material.