Analysis of the NMNAT2 interactome reveals that Fbxo21 regulates Wallerian degeneration. (A) A schematic diagram of the MS–based NMNAT2 interactome analysis. (B) Coomassie blue staining of the immunoprecipitates from the N2a cells expressing NMNAT2-FLAG using an anti-FLAG antibody. Three biological repeats (R) are shown. A protein band at slightly above 70 kDa detected in the NMNAT2-FLAG groups only (white box) is excised for in-gel digestion and the subsequent LC-MS/MS analysis. HC, heavy chain. (C) A volcano plot showing the proteins in the cut band in (B) identified by MS. Red dots, 107 proteins associated with NMNAT2-FLAG (fold change >2, P value <0.05). (D) A list of E3 ligase proteins identified in the NMNAT2 interactome. (E) A schematic of the in vitro drop cultures of primary mouse DRG neurons and the axotomy model. (F–I) Representative phase contrast images (F–H) and quantification (I) of injury-induced axon degeneration of primary mouse DRG neurons. Images are captured live at indicated time points after axotomy. (J) Western blot analysis confirming the KD of Fbxo21 by sh-Fbox21 and the expression of the RR Myc-Fbxo21^RR in the DRG cultures treated with sh-Fbox21. sh-Ctrl, scrambled shRNA. % of deg. axons, percentage of degenerated axons (see Materials and methods). Data are shown as mean ± SEM. n = 7–8 DRG cultures per group from pooled results of three independent repeats. The statistical significance is determined by two-way ANOVA; *P < 0.05 and ***P < 0.001. Scale bar: 50 µm. Source data are available for this figure: SourceData F1.