Figure S2.

Identification of Anpep as a target for the 4e8 antibody. (A) Coomassie-stained SDS-PAGE gel of eluates from immunoprecipitation with negative control (-con; lysates incubated with Protein G agarose beads only), anti-4e8, or anti-2h9 (a different mouse monoclonal antibody used for comparison) from 5-dpf larvae. The ~150-kD band (arrow) in the Coomassie-stained gel in the 4e8 IP lane was excised and analyzed by MS. (B) Mapped MS peptides (yellow) with zebrafish Anpep (A0A0R4IZV6_DANRE; anpepb, ENSDARG00000103878). Proteins were filtered at a false discovery rate <1%, with a minimum of four mapped peptides. Hits were sorted by the number of mapped exclusive unique peptide counts, leaving Anpep as the top hit with 44 exclusive unique peptides and 59% coverage. The observed molecular weight for the band in A is consistent with that of human ANPEP due to N- and O-glycosylation modifications (Naim et al., 1999).