Effect of Numb or Numb/NumbL cKO on contractile function of EDL muscle during ex- vivo physiological testing. (A) Specific tension generated during tetanic contraction at the indicated frequencies is shown for weight normalized EDL muscle harvested from HSA- MCM/Numb(f/f)) or HSA-MCM/Numb(f/f/NumbL(f/f) mice at 14-days after starting injections of tamoxifen or vehicle. Statistical analysis was performed with repeated measure ANOVA followed by Sidak’s multiple comparison test. F = 28.29, DFn= 6, DFd= 22; ***p< 0.001, force X frequency interaction ***p< 0.001; (B) Maximum force generated during a single twitch is shown. Statistical analysis was performed with one-way ANOVA with Tukey’s post-hoc test (F=10.07, DFn=3, DFd=13). *p<0.05; **p<0.01. N=3-6

Time to peak tension (A), half-relaxation time (B), and fatigue index (C) are shown for HSA-MCM/Numb(f/f)) and double (HSA-MCM/Numb(f/f/NumbL(f/f) mice) at 14 days after starting injections of tamoxifen or vehicle. Data were analyzed by one-way ANOVA with Tukey post-hoc test. (A) F=4.561, DFn=3, DFd=13; *p<0.05; (B) F=3.679, DFn=3,DFd=13 ; (C) F=0.9772, DFn=3, DFd=13. N=3-6.

Numb-interacting proteins identified by mass spectrometry.

A Western blot showing a representative co-immunoprecipitation of Septin7 by anti- Numb antibody. IgG: immunoprecipitation with control Ig; N: immunoprecipitation with anti-Numb; input: original C2C12 lysate. The upper panel was probed with anti-Septin 7, the lower panel with anti-Numb. B-D. Immunolocalization of Numb and Septin 7 in muscle. Longitudinal cryosections of TA muscle from C57Bl6 mice were immunostained for Numb and Septin 7: (B) merged image; (C) anti-Numb antibody (green); (D) anti-Septin 7 antibody (red); (E) DAPI staining of nuclei (blue). Scale bar, 10 μm. The inset in A shows higher magnification (x2.3) of the area in the black square.

Septin 7 is enriched at neuromuscular junctions. A-B. Violin plots generated by Myoatlas showing abundance of Septin 7 (A) and Numb (B) mRNA in nuclei of TA muscle from 5-month old mice [30] C. Representative confocal microscopy image of the neuromuscular junction of an isolated myofiber immunostained with anti-Septin 7 (red) and with a fluorescently tagged α- bungarotoxin (green); nuclei were stained with DAPI (blue). Scale bar, 10 μm.

Representative confocal images of isolated mouse hindlimb fibers immunostained with anti-Numb (green) (B and E) and anti-Septin 7 antibodies (red) (C-F); merge: merged images (A and D). Nuclei were labeled with DAPI (blue). A-C: myofiber from a control mouse (vehicle treated); D-F, myofiber from a Numb cKO mouse (tamoxifen treated). Scale bar, 10 μm.