Figure 6.

Confirmation of the candidate ligand molecule for Siglec-7 using proximity labeling (PL). K562 cells were incubated with complexes of recombinant Fc fusion proteins (Fc, Sig7EcFc, Sig9EcFc) and anti-human IgG+M+A-HRP. The proteins adjacent to HRP molecules were labeled with hydrogen peroxide and biotinyl-tyramide as substrates. A, lysates were prepared, and total biotin-modified proteins were detected using western blotting with Streptavidin-HRP. Pull-down experiments of the proximity labeled lysates (200 μg) with Fc, Sig7EcFc, and Sig9EcFc were performed using streptavidin-Sepharose. The precipitates were detected with (B) streptavidin, (C) Sig7EcFc, or (D) anti-leukosialin antibody.