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Fig. 1. FTI treatment defines two cellular pools of RhoB. (A) (a) HeLa cells were allowed to accumulate fluorescent-labelled transferrin (red) for 1 hour and then fixed and stained for endogenous RhoB (green). (b) Cells were co-stained for endogenous RhoB and LAMP-1 (red). Endogenous RhoB did not colocalise with early or recycling endosomes as marked by transferrin (a) but showed clear colocalization with the late endosome/lysosome marker LAMP-1 (b). Scale bar, 10 µm. (B) Single confocal sections taken through the broadest part of the cells showed that endogenous RhoB (green) has a heterogenous distribution between endocytic and plasma membrane pools (c). FTI treatment caused a complete loss of the plasma membrane pool, with a corresponding gain in endocytic staining (d). Myc-epitope tagged RhoB (green) localised almost entirely to endosomal structures (e), a fraction of which became clustered near the nucleus with FTI treatment (f). Scale bars, 10 µm. (C) The relative distribution of RhoB between plasma membrane and endosomal pools was quantified from confocal sections of FTI-treated and untreated cells using Scion Image. The data represents the mean±s.e.m. of three independent experiments, where 10 c ells were quantified for each condition.
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