Figure 4

Uptake of Aβ(1–40), Aβ(1–42) and Trf in SH-SY5Y cells under conditions that perturb clathrin mediated endocytosis. (a) Uptake of HF488-labelled Aβ(1–40) and Aβ(1–42) in cells treated with CPZ (5 µg/ml). The peptide uptake is reported as mean cellular uptake relative to control (cells not treated with inhibitor) for 4 independent experiments, each performed in triplicate. Statistical analysis of the data performed by one-way ANOVA with matched data followed by multiple comparisons with Bonferroni post-hoc test gives adjusted p-values for the individual comparisons made as: Aβ(1–40) vs. control 0.0007; Aβ(1–42) vs. control <0.0001; Aβ(1–40) vs. Aβ(1–42) 0.0289. (b) Flow cytometry histogram of live cells 24 h post transfection with mRFP-labelled AP180-C: the cells were gated for peptide uptake based on transfection efficiency as measured by the intensity of the mRFP label. (c) Uptake of HF488-labelled Aβ(1–40), Aβ(1–42) or AF647-labelled Trf in AP180-C transfected cells, gated as in (b) and related to peptide uptake in cells gated as non-transfected (N = 2, n = 2–4). (d–f) Confocal imaging of AP180-C (red) transfected cells imaged 24 h post transfection and incubated with HF488-labelled (d) Aβ(1–40), (e) Aβ(1–42) or (f) AF488-labelled Trf (green). In all experiments the concentration of Aβ was 1 μM and the concentration of Trf was 5 µg/ml. Cells were incubated with Aβ for 1 h and Trf for 5 min (flow cytometry) or 10 min (confocal microscopy).