Fig. 2

Flotillins mediate transferrin sorting into a Rab5 to Rab11a recycling pathway. A Representative confocal images of transferrin-Alexa488 internalisation in WT (left panel) and FlotKO (right panel) Jurkat T cells expressing mCherry-Rab5. B Mean transferrin-Alexa488 intensity present in Rab5 endosomes in WT or FlotKO Jurkat T cells over time. Dotted and dashed lines represent the timepoints from which the transferrin-Alexa488 intensity significantly diverges in Rab5 endosomes between WT and FlotKO. Pooled Data from 15 (WT) and 13 (KO#1) imaged cells. C Transferrin-Alexa488 vesicle number over time. Dotted and dashed lines represent time of intensity divergence calculated in B. Pooled data from 12 (WT) and 10 (KO#1) imaged cells. D Representative confocal images of transferrin-Alexa488 internalisation in WT (left panel) and FlotKO (right panel) Jurkat T cells expressing mCherry-Rab11a. E Mean transferrin-Alexa488 intensity present in Rab11a endosomes in WT or FlotKO Jurkat T cells over time. Dotted line represents the timepoint from which the transferrin-Alexa488 intensity significantly diverges in Rab11a endosomes between WT and FlotKO. Pooled data from 9 (WT) and 15 (KO#1) imaged cells. F Mean transferrin-Alexa488 intensity present in Rab11a endosomes in WT or FlotKO Jurkat T cells during 10 min imaging. G Representative confocal images of WT (left) or FlotKO (right) Jurkat T cells incubated with transferrin-Alexa488 for 20 min prior to fixation. H Mean fluorescence dispersion of transferrin-Alexa488 in WT and FlotKO Jurkat T cells. I Representative TIRF time series of transferrin-positive vesicle transiently interacting with the flotillin-positive compartment. J Mean transferrin-Alexa488 intensity present in flotillin-positive compartment in Jurkat T cells during 2-min imaging. Data points indicate single cells (F, J) or means of independent experiments (H); error bars indicate mean ± SEM. **p < 0.01 from unpaired, two-tailed Student’s t-test. Dotted line = p < 0.05 and dashed line = p < 0.01 from Wilcoxon rank-sum test of pooled data. Scale bars = 5 μm