Fig. 5

Modulation of Fpn internalization by hepcidin and holo-Tf. HEK 293 cells were used to determine the dynamics of holo-Tf and hepcidin on Fpn internalization using PLA, reported as integrated density per cell in the field of view per image (A–D). Pretreatment with PYR-41 (C) prevented the hepcidin induced reduction of interaction between holo-Tf and Fpn (B). The isolation of membrane bound Fpn confirms that hepcidin and holo-Tf co-incubation greatly reduces membrane Fpn levels, and this is prevented with PYR-41 (E–F). Hepcidin reduces membrane Fpn at a faster rate than holo-Tf (G, H). n = 3 to 5 for all experiments, means of biological replicates ± SEM were evaluated for statistical significance using one- way ANOVA with Tukey’s posttest for significance (D) and (F) or two-way ANOVA with Sidak’s post-test for significance (H). *p < 0.05, ***p < 0.001