Fig. 7: Identification of the holo-Lf binding sites on APP required for holo-Lf-induced amyloidogenic processing of APP.

A APP peptide reactivity to holo-Lf (1 μg/ml; 2 h) was determined by visual observation (+++ strong, ++ moderate, + weak) and specificity of binding to holo-Lf by peptides 40, 48, 49, 50 and 55 was confirmed using apo-Lf and detection antibody treatment alone (see Supplementary Fig. S10A). B Model of holo-Lf binding sites (coloured as shown in (A)) overlaid on the APP-E2 structure [97]. C Potency of the holo-Lf binding peptides of APP was assessed by dose dependently pre-incubating the peptide with holo-Lf (500 nM; 2 h) in vitro before adding to wt-APP⁶⁹⁵ SH-SY5Y and evaluating sAPPβ secretion in the media after a further 2 h. wt-APP⁶⁹⁵ SH-SY5Y exposure of APP peptide alone at each respective concentration showed no change in sAPPβ levels (data not shown). IC₅₀ for each peptide is shown in (A). D Covering the main holo-Lf binding sites, APP peptide 40, 49 and 55 were used to evaluate combinatory inhibition of holo-Lf-induced Aβ production in wt-APP⁶⁹⁵ SH-SY5Y. As in (C), peptide 49 with 40 or 55 (5 and 10 µM) was pre-incubated with holo-Lf (500 nM; 2 h) before addition to neuronal media for a further 2 h. Data are mean ± SEM of two experiments performed in duplicate with statistical analysis by two-way ANOVA comparing holo-Lf treated control, ****p < 0.0001, 5 µM peptide 49 alone, ^^^^p < 0.0001 or 10 µM peptide 49 alone, ^####p < 0.0001.