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. 2012 Aug 9;287(40):33304–33313. doi: 10.1074/jbc.M112.395608

FIGURE 6.

FIGURE 6.

MALDI-MS spectra of secreted Aβ from APP751 wt and meprin β single and co-expressing cells. HEK293T cells were co-transfected with APP751 wt and meprin β and single-transfected as control. Additionally, co-expressing cells were treated with 5 μm DAPT, or DMSO overnight. With the monoclonal antibody 4G8, immunoprecipitated Aβ was analyzed using MALDI MS analysis. Shown are the mass spectra of one representative measurement of three independent experiments. A, mass spectrum showing Aβ1–40 peptides detected in the medium of APP751 wt single-transfected cells. B, in meprin β single transfected cells, no Aβ signal could be detected. C, after APP and meprin β co-transfection, in addition to the Aβ1–40 signal, an amino-terminal truncated Aβ2–40 peptide could be observed. D, both Aβ1–40 and Aβ2–40 production could be abolished using DAPT, a γ-secretase inhibitor. E, DMSO did not influence the production of both Aβ species. In another approach, PS70 cells stably expressing meprin β (or GFP as a control) were additionally infected with an APP cDNA containing adenovirus. Additionally, cells were treated with 5 μm DAPT (H), 1 μm BACE inhibitor IV (I), 100 μm actinonin (J), or DMSO (F and G) as a vehicle control overnight. With the monoclonal antibody W0–2, immunoprecipitated Aβ was analyzed using MALDI MS analysis. Shown are the mass spectra of Aβ peptides of the different treated cells. F, in cells expressing APP and GFP, the mass spectrum shows Aβ1–40, Aβ1–39, representing a normal Aβ pattern. G, in cells expressing APP and meprin β, the Aβ1–40 peak increased and an additional peak representing Aβ2–40 appears due to meprin β cleavage. H, after DAPT treatment of cells co-expressing APP and meprin β, no Aβ signal could be detected, suggesting that also meprin β cleaved Aβ is dependent on γ-secretase cleavage. I, treatment of these cells with actinonin, an inhibitor for meprin β, resulted in a loss of the Aβ2–40 species, proofing the specificity of meprin β toward the Aβ sequence. J, treatment with BACE inhibitor IV did not abolish the Aβ1–40 or the Aβ2–40 signal (n = 1). Theoretical and observed masses for Aβ1–40 and Aβ2–40 are 4328.2/4328.3 and 4213.1/4213.3 Da, respectively. Aβ2–40 was corroborated by LIFT sequencing.