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. 2013 Dec 28;289(8):5109–5121. doi: 10.1074/jbc.M113.510131

FIGURE 2.

FIGURE 2.

Aβ and AICD species generated by the reconstituted lipid raft Aβ generation system. Lipid rafts prepared from CHO cells were subjected to the reconstituted Aβ generation assay. Following incubation for the indicated times, the reaction mixtures were subjected to either Western blotting or IP/TOF mass spectrometry. A, Western blotting with 82E1 (total Aβ) and Aβ40- and Aβ42-specific antibodies. Aβ peptide standards were applied onto each rightmost lane. B and C, quantification of the signal intensities for total Aβ (closed circles in B), Aβ40 (closed squares in C), and Aβ42 (open triangles in C) on the blots, which were expressed as picomoles of Aβ generated per ml of the reaction mixture. Data are represented as means ± S.E. from four independent experiments. D, Western blotting with 82E1 using a long SDS-urea gel. Authentic Aβ species from Aβ37 to Aβ49 were loaded onto the rightmost three lanes (M1: Aβ37, Aβ38, Aβ40, Aβ42, Aβ45, and Aβ46 to Aβ49; M2: Aβ39, Aβ41, and Aβ44). In this gel system, Aβ46 and longer Aβ species (Aβ47 to Aβ49) were not separated. Aβ40 was robustly produced by lipid rafts. Aβ38, Aβ42, Aβ43, Aβ45, and Aβ46 and longer species were also produced in a time-dependent manner. Asterisks in D indicate C99-FLAG or its carboxyl-terminally truncated fragments. E, IP/TOF-mass spectrometry for shorter Aβ species. An aliquot of the reaction mixture was subjected to Western blotting with 82E1 (upper panel). Aβ in the reaction mixture was immunoprecipitated with 6E10 and subjected to TOF-mass spectrometry (lower panel). Among the candidate Aβs, only Aβ(1–16) and Aβ(1–15) were identified after incubation. However, L-685,458 at 2 μm suppressed Aβ generation almost completely (upper panel) but not generation of the shorter Aβs, indicating that the generation of Aβ(1–16) and Aβ(1–15) was not mediated by γ-secretase (26). Aβ(1–17) and longer species were undetectable in this system. F, IP/TOF-mass spectrometry for AICDs. An aliquot of the reaction mixture was subjected to Western blotting with FLAG antibody (upper panel). AICD produced was subjected to IP/TOF mass spectrometry (lower panel). The signals corresponding to AICD(50–99) and AICD(49–99) were identified after incubation. They were suppressed by L-685,458 at 2 μm, indicating γ-secretase-mediated generation.