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Fig. 1 | BMC Biology

Fig. 1

From: Rapid increase in transferrin receptor recycling promotes adhesion during T cell activation

Fig. 1

T cell activation stimulates uptake and recycling of transferrin and TfR. A Transferrin uptake stimulation upon T cell activation. Jurkat T cells were equilibrated at 37 °C in the presence of transferrin-Alexa647 and subsequently left untouched (resting), or 30 min treated with non-stimulating solution (0 min activated) or 30 min stimulated with activating antibodies anti-CD3ε + anti-CD28. Uptake was stopped on ice and cells were analysed for transferrin-Alexa647 uptake. Displayed is the fold-change of Alexa647 signal relative to untouched (resting) controls. B Transiently boosted TfR recycling upon T cell activation. Jurkat T 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 upon activation, cells were activated with soluble activating 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 the corresponding resting cells. C Increased surface TfR levels upon T cell activation correlates with boosted TfR recycling. Jurkat T cells were activated with soluble activating antibodies (anti-CD3ε + anti-CD28) or left untreated for the indicated times and immediately fixed with 3.7% paraformaldehyde (PFA). Surface TfR was then stained with biotinylated anti-TfR, which was detected with streptavidin-Pacific Blue. Data points and error bars indicate mean of n = 4 independent experiments and standard error of the mean (SEM). D Increased surface TfR levels upon activation of expanded primary human T cells. Expanded primary T cells were activated with soluble activating antibodies (anti-CD3ε + anti-CD28) or left untreated for the indicated times and immediately fixed with 3.7% PFA. Surface TfR was then stained with biotinylated anti-TfR, which was detected with streptavidin-Pacific Blue. Data points indicate mean of n = 3 independent experiments ± SEM. E Increased TfR recycling upon T cell activation. Expanded primary human T cells were treated as described in B. Results were analysed and depicted as in B. Statistical significance determined with unpaired two-tailed Student’s t-test (A), one-way ANOVA (B, E) or two-way ANOVA (C, D). **p < 0.01; ****p ≤ 0.0001, no indication—not significant

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