Extended Data Figure 3: Decreased phosphorylation of NF-M and NF-H upon sustained Stmn2 suppression.

(a) Representative micrographs of motor roots and higher magnification images of ventral root motor axon morphology and diameters, 8 months after subpial injection of AAV9 encoding non-targeting or Stmn2 targeting shRNA. (b,c) Quantification of cross-sectional area (n=5 animals/condition) (b) and of the total number of axons per ventral root (n=5 animals/condition) (c). Statistical analysis by two-sided, Mann Whitney t-test, (P = 0.0079). (d,e) Levels of total neurofilament heavy (NF-H) and neurofilament light (NF-L) (f) and total neurofilament medium (NF-M) (g) analyzed by immunoblotting spinal cord extracts from WT mice 8 months after subpial injection of either AAV9 encoding a non-targeting (n=6 animals) or Stmn2 targeting shRNA (n=5 animals). β3-tubulin was used as loading control. AAV9-shRNA-mediated suppression of stathmin-2 protein levels was confirmed in all examined samples (e). (f-h) Quantification of the immunoblots in panels d,e. Statistics by two-sided, unpaired t-test. (i) Levels of phosphorylated neurofilament heavy (pNF-H) and medium (pNF-M) subunits analyzed by immunoblotting of spinal cord extracts from WT mice, 8 months after subpial AAV9 encoding a non-targeting or Stmn2 targeting shRNA. β3-tubulin was used as a loading control. (j,k) Quantification of the immunoblots from panel i. N=5 animals with AAV9-shControl and n=6 animals with AAV9-shStmn2 (j), and n=13 animals with AAV9-shControl and n=12 animals with AAV9-shStmn2, (P = 0.036) (k). Statistics by two-sided, unpaired t-test. All panels: Each data point represents an individual mouse. Error bars plotted as SEM. ****, P <0.0001; ***, P < 0.001; **, P < 0.01; *, P <0.05; ns, P >0.05.