Fig. 1

Modulation of Fpn protein levels in ECs by holo-Tf. iPSC-derived ECs were cultured on bi-chamber plates, incubated with apo- or holo-Tf in the basal chamber, and collected after 8 h for immunoblotting. Fpn protein levels were normalized to cyclophilin B as a loading control. All quantifications were further normalized to untreated control to account for cell count variability. Holo-Tf decreased Fpn protein levels by 50% at concentrations as low as 0.1 μM, while apo-Tf did not (A–C). Holo-Tf-mediated internalization and degradation of Fpn was inhibited by a ubiquitination inhibitor, PYR-41, (D–F) confirming that holo-Tf’s decreases Fpn through the established degradation pathway. ECs were incubated with 0.25 μM holo-Tf to observe the ubiquitination and internalization of Fpn over time. After 1 h, ubiquitination of Fpn was detected, with a maximal effect at 3 h (G–J). By 5 h, 50% of Fpn is internalized with continuous ubiquitination per Fpn present. n = 3 for all experiments, means of biological replicates ± SEM were evaluated for statistical significance using one- way ANOVA with Tukey’s posttest for significance. *p < 0.05, ***p < 0.001