Figure 7.

Effect of CMT2B-associated Rab7 mutations on EGF degradation. HeLa cells were transfected with GFP-tagged Rab7 wt, Rab7 T22N, Rab7 Q67L, Rab7 L129F, Rab7 N161T, Rab7 V162M, or nontransfected (NT) as indicated. Then cells were incubated 1 h at 4°C with rhodamine-labeled EGF, washed, and incubated at 37°C for 15 min or 3 h. Finally, they were processed for confocal fluorescent microscopy. Transfected cells were selected, and the total red signal in the entire cell (representing undegraded EGF) was quantified. Staining of EGF at 15 and 180 min is shown. Arrows indicate transfected cells. Intensities of EGF were quantified and plotted as percentage of the respective intensities after 15 min incubation at 37°C (bottom right). Values are calculated on 30 cells for each construct and represent means ± SEM. Differences between the values of Rab7 CMT2B mutated proteins and that of Rab7 T22N are statistically significant (p value <0.01) at 180 min. Differences between the values of Rab7 V162M and those of Rab7 wt or the other Rab7 CMT2B mutated proteins are not statistically significant.