Fig. 3. Holo-Lf-mediated APP internalisation is clathrin independent and ARF6 dependent.

A, B Cell-surface APP (ab15272) response to holo-Lf (500 nM; 2 h) as monitored by FACS in non-permeabilised SH-SY5Ys after control non-targeted of CHC knockdown by RNAi (40 nM; 72 h) (A) or ARF6 RNAi (20 nM; 48 h) (B). C, D Deconvoluted images of double immunofluorescence confocal microscopy of wt-APP⁶⁹⁵ SH-SY5Ys treated with holo-Lf. After RNAi depletion of CHC or ARF6 as in (A, B), surface APP was labelled with an APP antibody (22C11) (green) at 4 °C before replacing media with holo-Lf (1 μM; 1 h) at 37 °C. An APP secondary detection antibody was then added with total CHC (ab21679) (red) (C) or ARF6 (ab131261) (red) (D) co-labelling. Co-localisation of APP with CHC (C) and ARF6 (D) is represented as yellow in the merged image (white arrows). E, F Biotinylated holo-Lf (0.5 mg/ml; 1 h at 37 °C) added to SH-SY5Ys transfected with control non-target and CHC (40 nM; 72 h) (E) or ARF6 ± APP RNAi (20 nM; 48 h) (F) before being subjected to 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 with streptavidin-HRP. Data are mean ± SEM of three experiments performed at least in duplicate. Statistical analysis in A, B was by two-way ANOVA or two-tailed t-tests for E, F, ****p < 0.0001 depicts fold change compared with levels derived from non-targeting control, ^^^^p < 0.0001 compared to CHC RNAi (A) or holo-Lf-treated (B) non-target control and ^##p < 0.0001 compared to ARF6 RNAi (B). C, D Images are a representative from multiple cells within experiments carried out in duplicate. Scale bar = 10 µm.