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. 2015 Aug 18;1:15021. doi: 10.1038/celldisc.2015.21

Figure 5.

Figure 5

Photoclickable analog XYT1032 reveals XYT472B and 3AA binding sites on PS1. (a) Chemical structure of XYT1032. (b and c) XYT1032 reduces PS1-NTF/BACE1 interaction in split-TEV assay (b) and total Aβ production (c). Transfected HEK293MSR cells were treated with chemicals for 16 h before DLR analysis of luciferase reporter activities, and HEK293/APPswe cells were treated for 8 h before the supernatants were collected for ELISA. (d) Labeling and affinity purification of γ-secretase subunits and BACE1 by XYT1032 in different detergents. Cells expressing PS1, Pen2, NCT, C-terminal Flag-tagged APH1aL and BACE1 were treated with XYT1032 (3 μM) and photo-activated click reaction were carried out. γ-secretase subunits and BACE1 were affinity purified by anti-Flag or straptavidin resin as indicated. (e) XYT1032 binds only to PS1. Cells expressing β-Gal, C-terminal Flag-tagged APH1aL, NCT, PS1 (FL), Pen2, BACE1 or PS1-NTF, respectively, were treated with XYT1032 (3 μM) and subjected to photo-activated click reaction and affinity purification. (f) XYT472B and 3AA compete with XYT1032 in labeling of PS1-NTF. Cells were treated with 3 μM XYT1032 in the absence or presence of 10 μM, 30 μM, 100 μM XYT472B or 3AA for 2 h. (g) Secretase inhibitors and modulator fail to compete with XYT1032 in binding to PS1-NTF. Cells were incubated with 3 μM XYT1032 in the absence or presence of 100 μM designated chemicals. (h) XYT472B and 3AA enhanced the labeling of PS1-NTF by E2012-BPyne. Cells were incubated with 1 μM E2012-BPyne in the absence or presence of 50 μM designated chemicals for 1 h. After chemical incubation, photo-activated crosslinking, click reaction and streptavidin affinity purification in 1% CHAPSO buffer were performed. C, CHAPSO; F, Flag; S, streptavidin; T, TritonX-100.