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Fig. 7. Endocytosis of TxR-Tf is inhibited in cells expressing GFP-Rab21 T33N. Hep3B cells transfected with GFP-Rab21 plasmids were incubated for 15 minutes at 37°C with 50 nM TxR-Tf, washed on ice, fixed and analysed by fluorescence microscopy. There was no visible difference in the levels or distribution pattern of internalised TxR-Tf (red in merge) in nontransfected cells and those expressing GFP-Rab21 wt (A-C) or GFP-Rab21 Q78L (D-F). Levels of internalised Tf were significantly lower in cells expressing GFP-Rab21 T33N and the label was confined to relatively small vesicles (G-I). Arrows in A-C point to Rab21 wt colocalisation with internalised TxR-Tf. (J-M) HeLa cells transfected with GFP-Rab21 wt plasmid were incubated for 4 minutes at 37°C with 50 nM TxR-Tf, washed on ice, fixed and labelled with anti-EEA1-and Alexa647-conjugated secondary antibodies, followed by confocal fluorescence microscopy. Arrows point to Rab21 wt, EEA1 and TxR-Tf colocalising structures. Asterisks represent transfected cells. Bars, 10 µm. (N) HeLa cells transfected with GFP-Rab21 wt plasmids were incubated with 50 nM TxR-Tf, for either 4 minutes at 37°C, 1 hour at 16°C or for 30 minutes at 37°C followed by washing and chasing for a further 30 minutes at 37°C in the absence of TxR-Tf, before fixing and analysis by fluorescence microscopy. Quantification of the number of TxR-Tf structures colocalising with Rab21 was determined for the different internalisation conditions. Error bars show mean and standard deviation between individual cells. Asterisk indicates statistical significance of P<0.001 compared with 4 minutes internalisation at 37°C.
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