The chase media from microglia (A; chase day 2), J774 cells (B; chase day 1), and J774 cells (C; chase day 2) that were pulsed with fAβ peptides overnight were collected and subjected to TCA precipitation. The TCA insoluble material was solubilized in 70% formic acid, and immunoprecipitated using monoclonal Aβ antibody 4G8. The eluted samples were measured using mass spectrometry. The identities of the observed peaks are indicated using human Aβ sequence numbers. Representative spectra from ten experiments are shown. * indicates the protonated ion peak; ** is the mono-sodium adduct peak.

A). Microglia [(i) chase day 0 (ii) chase day 1 and (iii) chase day 2] B) J774 macrophages [(i) chase day 0 (ii) chase day 1 and (iii) chase day 2] were pulsed with fAβ peptides overnight. The cells were then rinsed and chased for indicated times. At the end of each chase time, the cells were rinsed and lysed in lysis buffer. The cells were sonicated and centrifuged at 100,000 × g for 1 h at 4°C. The resulting pellet was solubilized in 70% formic acid, followed by immunoprecipitation using monoclonal Aβ antibody 4G8. The molecular masses of eluted samples were measured using mass spectrometry. The identities of the observed peaks are indicated using human Aβ sequence numbers. Representative spectra from microglia and J774 cells from ten experiments of each cell type are shown. * indicates the protonated ion peak; ** indicates the mono-sodium adduct peak; *** indicates the disodium adduct peak.

Cells were incubated for 45 min with Cy3-fAβ42 (1 µg/ml). After incubation the cells were rinsed extensively with complete media and were chased in (A) complete media (B) complete media+30nM mannose-6 phosphate phosphorylated lysosomal enzyme (C) complete media +30nM mannose-6 phosphate phosphorylated lysosomal enzyme + 10mM mannose-6 phosphate. After 3 days, the cells were rinsed extensively, fixed, and imaged by digital fluorescence microscopy. The integrated Cy3 fluorescence power per cell before and after the chase was quantified. The integrated cell-associated fluorescence power (arbitrary units) after 3 days normalized to the fluorescence power at day 0 is shown in the figure. The P value (unpaired Student’s t test) for fAβ degradation by microglia after add-back of 30nM mannose-6 phosphate phosphorylated lysosomal enzyme was less than 0.005 compared to either control microglia or enzyme addition in the presence of 10mM mannose-6 phosphate. Data for each condition show the average of the fluorescence power values from 3 different experiments done on 3 different days. Error bars, represent S.E.

(A) ¹²⁵I-labeled fAβ. Microglia, J774 cells and primary mice peritoneal macrophages were incubated for 1 h with ¹²⁵I-labeled fAβ 1 µg/ml) after which the cells were washed and chased for varying times. In parallel samples, excess Ac-LDL (200 µg/ml) or fucoidan (500 µg/ml) was added to the radiolabeled fAβ to determine the extent of nonspecific cell-associated radiolabeled fAβ, for which all measurements were corrected. At each chase time, the chase medium was collected, and cells were solubilized. The chase medium was subjected to TCA precipitation to monitor degradation. The radioactivity of TCA-soluble fractions in peritoneal macrophages (Δ), J774 cells (●), and microglia (■) are shown as the % of total radioactivity. The data presented are averages of the radioactive counts from three dishes per condition from six different experiments using fAβ42. Error bars, S.E.
(B) Cy3-fAβ. Cells were incubated for 45 min with Cy3-fAβ42 (1 µg/ml) and chased for various times. After each chase time, the cells were rinsed extensively, fixed and imaged by digital fluorescence microscopy. The Cy3 fluorescence intensity remaining in each cell over the course of the chase was quantified. The integrated cell-associated fluorescence power (arbitrary units) normalized to the fluorescence power at day 0 over the course of 3days is shown in the figure. Data for each time point shows the average of the normalized fluorescence power values from 3 different experiments done on 3 different days. Error bars, represent S.E.