Fbxo21 regulates the protein turnover of NMNAT2 in DRG neurons. (A) A schematic of the in vitro drop cultures of primary mouse DRG neurons and the pulse chase assays. Whole cell cultures (soma and neurites) or neurite-only samples are collected as indicated. (B–G) Representative western blot images (B, D, and F) and quantifications (C, E, and G) of NMNAT2 protein levels in soma and neurites (B and C) or neurites only (D–G) of the mouse DRG cultures infected with scrambled shRNA(sh-Ctrl) or sh-Fbxo21 at the indicated time points after the CHX treatment (B–E) or axotomy (F and G). (H and I) For cross comparison, the NMNAT2 turnover curves of the sh-Ctrl groups in C, E, and G are replotted in H and those with sh-Fbxo21 in I. (J–L) NMNAT2 turnover in DRG axons treated with CHX, axotomy, or CHX + axotomy (J) is assessed by western blots analysis (K and L). All NMNAT2 levels are normalized to Tuj1, and the relative level of each group at 0 h is set to 100%. Mean ± SEM. n = 3–5. Two-way ANOVA; *P < 0.05 and **P < 0.01; ns, not significant. Source data are available for this figure: SourceData F3.