FBXO45 has shared function with SPSB1/SPSB2 as a NOS2-interacting protein. (A) Putative NOS2 complex containing the ECS(SPSB) versus SCF(FBX045) E3 ligases. Shaded proteins were identified in NOS2 co-IPs. (B) Alignment of conserved B30.2/SPRY domains of human FBXO45, SPSB1 and SPSB2. Residues implicated in substrate interaction are highlighted in yellow. (C) 5 μg each of plasmids expressing wt-NOS2-Flag and myc-FBXO45 were transiently transfected separately, or in combination, in HEK-293 cells. After 24 h, total cell lysates were co-IP’d overnight with either M2-Flag or c-Myc agarose, and eluents were analyzed by western blotting. (D) Wild-type or N27A NOS2-Flag was co-transfected with myc-FBXO45 and interactions were analyzed by forward and reverse IP as in (C).