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. Author manuscript; available in PMC: 2017 May 18.
Published in final edited form as: ACS Chem Biol. 2015 Jun 10;10(8):1925–1931. doi: 10.1021/acschembio.5b00321

Figure 2.

Figure 2

L458-based photoreactive probes specifically label PS1 and SPP. (A) Structures of L458 and photoreactive probes JC8, L646, GY4, and L505. L458 side chain residues (P and P′ sites) interact with corresponding subpockets of γ-secretase and SPP (S and S′ sites). Photoreactive probes JC8, L646, GY4, and L505 have an L458 backbone (black), a biotin moiety (green), and a cross-linkable benzophenone (BP). Each probe has a BP incorporated into a different site on the L458 backbone. The location of the BP is illustrated by the color scheme. For example, JC8 has a BP at the P1′ site, in place of the red benzene ring. JC8, L646, GY4, and L505 label S1′, S2, S1, and S3′ subsites of the enzymes, respectively. (B) HeLa membranes were labeled with 20 nM of photoprobes JC8, L646, GY4, or L505 in the presence of 0.25% CHAPSO, and either with (+) or without (−) 2 μM L458. Samples were run on SDS-PAGE and analyzed by Western blot. Anti-SPP and PS1-NTF antibodies were used to detect SPP (upper panel) and PS1-NTF (lower panel). (C) Same as B, except 2 μM (Z-LL)2-ketone was used to block the labeling of SPP (upper panel) and PS1-NTF (lower panel).