Figure 5
From: Pharmacological targeting of the β-amyloid precursor protein intracellular domain
Photoaffinity labeling analysis of specific APP fragments.
(a) Schematic representation of APP processing products, including the binding sites of the antibodies utilized in this work and the APP fragments that were subjected to photolabeling analysis: the CHF5074-BpB-reactive CTFα and AICD-59 fragments (black bars) and the non-reactive Aβ42 peptide (white bar). (b) Photoaffinity labeling profiles (upper panels) obtained after incubation with (+) or without (−) CHF5074-BpB (3 μM) of the synthetic AICD-59 (20 pmol, left panel) and Aβ42 (20 and 90 pmol, central panel) peptides and the empty glutathione S-transferase carrier (GST) and the recombinant GST-CTFα fusion polypeptide (20 pmol, right panel). CHF5074-BpB reactive products were detected with IrDye 680-conjugated streptavidin. Labeling competition experiments were performed by preincubating 20 pmol of the AICD-59 peptide (left panel) or the GST-CTFα fusion polypeptide (right panel) in the presence (+) of unmodified CHF5074 (100 μM), followed by exposure to 3 μM CHF5074-BpB. Parallel immunoblot analyses performed with the anti-APP C-terminal antibody (AICD-59), the 6E10 antibody (Aβ42) and with an antibody directed against GST (GST and GST-CTFα), served as controls to verify the input of the various (poly)peptides utilized for these experiments (lower panels). Cropped gel images are shown and the gels were run under the same experimental conditions.
