Fig. 1. Characterisation of GFP-Rab21 variants. (A-C) Lysates from HeLa cells expressing GFP-Rab21 wt, GFP-Rab21 T33N, GFP-Rab21 Q78L or GFP alone were immunoprecipitated using anti-GFP antibodies and subjected to SDS-PAGE and western analysis before detection of GFP by ECL (A) or GTP binding using ³²P-GTP (B). Graph in C shows quantification of radioactivity from GFP-Rab21 bands in B and shows maximum GTP binding by GFP-Rab21 Q78L and baseline GTP binding by GFP-Rab21 T33N. (D-F) HeLa cells transfected using calcium phosphate with GFP-Rab21 wt (D), GFP-Rab21 T33N (E) and GFP-Rab21 Q78L (F) plasmids were fixed and processed for fluorescence microscopy. GFP-Rab21 wt and Rab21 Q78L predominantly label the perinuclear region, in addition to peripheral vesicular structures. Rab21 Q78L is also localised to a reticular network emanating from the perinuclear area to the cell periphery. Rab21 T33N labels the perinuclear region in addition to small cytoplasmic vesicles (E). Bars, 10 µm. Video microscopy of HeLa cells expressing GFP-Rab21 variants can be seen as QuickTime Movies 1-3 in supplementary material.