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Fig. 2. Subcellular localisation of GFP-Rab21. HeLa cells, transfected using calcium phosphate with GFP-Rab21 wt (A-C,G-O) or GFP-Rab21 T33N (D-F) plasmids were either directly fixed and labelled with anti-p230 (A-F), anti-Lamp-1 (G-I) and anti-transferrin receptor (J-L) antibodies, followed by rhodamine-conjugated secondary antibodies (red in merge), or were first incubated for 6 minutes at 37°C with 75 ng/ml Rh-EGF (M-O) before fixation. Confocal fluorescence microscopy shows some colocalisation between Rab21 wt and p230 in the more intensely labelled perinuclear area but GFP-Rab21 peripheral structures are free of this marker (A-C, arrowheads). Rab21 T33N overlaps extensively with p230 (D-F). There is very little overlap between Rab21 and Lamp-1 (G-I) but some overlap in peripheral structures with the transferrin receptor (J-L) and internalised Rh-EGF (M-O). Arrows indicate colocalising structures, and arrowheads indicate structures containing only Rab21. Bars, 10 µm.
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