Fig. 4: Holo-Lf promotes APP trafficking through the Rab11-positive recycling endosome.

A–C Flow cytometric quantification of cell-surface APP levels (ab15272) on the cell surface of non-permeabilised SH-SY5Ys with and without holo-Lf (500 nM; 2 h) after treatment with RNAi (20 nM; 48 h) for Rab5a (A), Rab7a (B) Rab11a (C) and a non-targeted control. D–F Within the same experimental parameters as A–C, quantification of the effect of Rab5a (D), Rab7a (E) and Rab11a (F) knockdown on internalisation of biotinylated holo-Lf (0.5 mg/ml; 1 h at 37 °C) was measured by the ligand internalisation assay. Residual surface biotin was stripped with MeSNa so that only internalised biotinylated Lf could be detected in the total cell lysate when analysed by western blot (shown in Supplementary Fig. S6). G Representative deconvoluted images from double immunofluorescence confocal microscopy of wt-APP⁶⁹⁵ SH-SY5Ys reverse transfected with RNAi for control non-target, Rab4a (i) or Rab11a (ii). In double knockdown, cells were reverse transfected with Rab4a and then forward transfected with Rab11a (iii) RNAi (20 nM; 48 h). After surface labelling with anti-APP (22C11) (green) at 4 °C, cells were treated with holo-Lf (1 μM; 1 h at 37 °C) and then permeabilised to label with the antibody to APP (green) and anti-Rab4 (ab13252; red) (i, iii) or anti-Rab11 (ab3612; red) (ii, iii). A–F Data are mean ± SEM of three experiments performed at least in duplicate. Statistical analysis by two-way ANOVA (A–C) or two-tailed t-tests (D–F), **p < 0.01, ***p < 0.001 and ****p < 0.0001 depict fold change compared to the untreated non-targeting control and ^^^^p < 0.0001 compared to Rab RNAi without holo-Lf. G Images are a representative from multiple cells within experiments carried out in duplicate. Scale bar = 10 µm.