Fig. 3

TfR surface delivery and boosted recycling relies on flotillins. A Representative TIRF images of transferrin-Alexa488 (cyan) vesicle co-fusion events with mCherry Rab11a (magenta) in WT Jurkat T cells (top) or fusion with (middle) or without (bottom) mCherry-Rab11a in FlotKO Jurkat T cells. B Quantification of the percentage of transferrin-Alexa488 fusion events containing mCherry-Rab11a in WT and two FlotKO Jurkat T cell lines. Fusion events were counted as containing the indicated protein if a decrease in maximum intensity from the last three frames was greater than 10% compared to the maximum intensity of the initial frame. Data points represent means of independent experiments. C Quantification of TfR recycling boost in WT and FlotKO Jurkat T cells upon activation (left) and representative histograms (right) of n = 4 independent experiments. Cells were incubated with biotinylated anti-TfR for 90 min to label all TfR pools with antibodies. Then, surface-exposed anti-TfR-biotin was blocked with unlabelled streptavidin. To assess TfR recycling, cells were activated with soluble antibodies (anti-CD3ε + anti-CD28) or left untreated for the indicated times. Antibody-labelled TfR delivered to the surface was detected with Pacific Blue-labelled streptavidin. Fold change of bound streptavidin-Pacific Blue in activated T cells is depicted relative to resting cells at timepoint 0 min. D Increased surface TfR levels upon T cell activation in WT vs FlotKO Jurkat T cells. Quantification (left) and representative histograms (right) of n = 3 independent experiments. Cells were activated with soluble antibodies (anti-CD3ε + anti-CD28) or left untreated for the indicated times. Surface TfR was then stained with transferrin-Alexa647. Statistical significance determined with one-way ANOVA (B) or two-way ANOVA (C, D).*p ≤ 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, no indication—not significant