The F-box domain of FBXO21 mediates the binding to NMNAT2 and is required for the assembly of the SCF ^FBXO21 complex. (A) The schematic diagrams of the FL and different truncation mutants of the FBXO21 proteins in B. (B) Representative image of the co-IP and western blot assays examining the interaction between NMNAT2 and the FL or different truncated FBXO21 proteins in 293T cells. ^#indicates the leftover signal of the band of NMNAT2-HA from anti-HA after stripping and reprobing the blots. (C and D) Representative western blot image (C) and a schematic diagram (D) showing that FBXO21 with deletion of either the N terminus (aa 1–29, ΔN) or the F-box domain (aa 30–78, ΔF-box) can still interact with NMNAT2, while deletion of them both (Δ1–78) markedly diminishes the binding in the co-IP experiment. (E) The endogenous mouse proteins Fbxo21, Skp1, and Cul1 are co-immunoprecipitated with NMNAT2-FLAG in N2a cells, and KD of Fbxo21 substantially reduces the co-IP of Skp1 and Cul1. (F and G) Representative western blot image (F) and a schematic diagram (G) showing that ΔF-box eliminates the binding of FBXO21 to SKP1, CUl1, and RBX1. Source data are available for this figure: SourceData F6.