Affinity purification mass spectrometry identifies MIB1 as a novel interaction partner with RYK. (A) Illustration depicts the RYK-associated protein interaction network discovered by affinity purification mass spectrometry (black lines). The STRING database was used to curate additional interactions among proteins found to interact with RYK (gray lines). The size of the node indicates the total number of peptides identified in eight independent purifications. (B) Schematic representation of RYK and MIB1 proteins depicting predicted domains. (C) Western blot for endogenous MIB1 demonstrates interaction with streptavidin affinity-purified glue-tagged RYK, RYKICD, or a membrane tethered form of the RYKICD (MYR-RYKICD). Untransfected, glue-TCF4, and glue-NCK2 cells served as negative controls. (D) Western blot for glue-RYKICD demonstrates interactions with flag affinity-purified full-length MIB1, the N terminus of MIB1, or a point mutant (C997S) of MIB1 that renders it catalytically impaired. N-terminally truncated MIB1 and negative controls do not copurify RYK. Blots represent n > 3.