(A) Left to right: immunoblot of low-level induced expression of FLAG-HA-ARIH1 or catalytic C375S mutant in 293 FLP-IN cells; high-confidence interacting proteins (HCIPs) for WT ARIH1 and ARIH1C357S, ± neddylation inhibitor MLN4924, identified by CompPASS (Sowa et al., 2009). ARIH1 predominantly interacts with CRL subunits colored by CRL1, blue; CRL2, yellow; CRL3, purple.
(B) TMT method to identify MLN4924-sensitive/neddylation-dependent ARIH1 HCIPs.
(C) ARIH1C357S HCIPs decreased ≥2-fold by neddylation inhibitor MLN4924 colored by CRL: CRL1, blue; CRL2, yellow; CRL3, violet. CRL3KLHL12 and substrate SEC13-SEC31A are highlighted. Error, ±SEM.
(D) Log2 fold change of ARIH1C357S interactors from the schematic in (C). Error bars, ±SE.