Figure 2.

A1 KO in microglia caused an increase in intracellular Aβ1–42 degradation in lysosomes. A, WT or A1 KO microglia were incubated with 0.5 μm Cy3-Aβ1–42 (red) for 3 h and immunostained with anti-LAMP1 antibody (green) for late endosomes/lysosomes. Cells were imaged using a confocal microscope. Representative immunofluorescence images are shown. Bottom, Enlarged images of the boxed areas in the top. Scale bar, 10 μm. B, Diagram demonstrating the procedure used to conduct pulse-chase experiments with or without various inhibitors. C, D, Pulse-chase experiments were performed in WT or A1 KO microglia, in a manner described in B and in Materials and Methods. Briefly, WT or A1 KO microglia were incubated with 0.5 μm Aβ1–42 for 30 min. Cells were preincubated with (D) or without (C) inhibitors as indicated for 90 min before exposure to oligomeric Aβ (Baf, bafilomycin A1; CatBi, cathepsin B inhibitor; PepA, pepstatin A methyl ester). Cells were washed several times and incubated in serum-free medium for the indicated time. At the end of incubation, cells were washed and lysed. Intracellular Aβ1–42 levels were examined by ELISA and normalized with cellular protein concentration. Data are mean ± SEM of three experiments. **p < 0.01. ***p < 0.001. ^##p < 0.01.