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Fig. 5. Endogenous Rab21 and GFP-Rab21 distribution in Hep3B cells. Hep3B cells were either directly fixed and stained with anti-Rab21 antibodies (A-C), or first transfected with GFP-Rab21 wt (D-F), GFP-Rab21 Q78L (G-I) or GFP-Rab21 T33N (J-L) plasmids using calcium phosphate before fixing and labelling with anti-EEA1 antibodies and Texas Red-conjugated secondary antibodies (red in merge) before analysis by confocal fluorescence microscopy. Endogenous Rab21 was seen to localise to punctate structures, many of which also contained EEA1 (A-C, arrows). In addition, many EEA1-positive, but Rab21-negative structures were observed (arrowheads). GFP-Rab21 wt expression results in the formation of swollen endosomes that label intensely with anti-EEA1 antibodies (D-F). GFP-Rab21 Q78L expression also causes slight enlargement of EEA1-positive endosomes (G-I). EEA1 labels smaller vesicles in nontransfected cells and those expressing Rab21 T33N (J-L). Arrows indicate colocalisation of GFP-Rab21 and EEA1, and asterisks represent transfected cells. Bars, 10 µm.

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