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. 2021 Jun 30;40(17):e107586. doi: 10.15252/embj.2020107586

Figure 5. CRMP4‐dynein interaction is enhanced in ALS motor neuron axons.

Figure 5

  1. Immunoprecipitation of DIC followed by Western blot analysis of CRMP4 in SOD1G93A compared to WT P90 sciatic nerves under physiological conditions. IgG antibody was used as a control.
  2. Quantification of the blot in A. 3 repeats, 12 sciatic nerves per condition were used in each repeat. The CRMP4 intensity band was normalized to the DIC intensity band in each repeat. Data presented as mean ± SE (Ratio Paired t‐test, *P = 0.0416).
  3. Immunoprecipitation of DIC followed by Western blot analysis of CRMP4 in COS7 cells that were transfected with mutant CRMP4 I141V compared to control. IgG antibody was used as a control.
  4. Quantification of four repeated pull down in C. The CRMP4 intensity band was normalized to the DIC intensity band for each repeat. (Student’s t‐test, n = 4, data presented as mean ± SE, *P = 0.0393).
  5. Representative images from the proximity ligation assay (For explanation of PLA technique; please refer to method section) for CRMP4 and dynein in SOD1G93A and WT primary MNs axons that were exposed to either control or Sema3A 8h post‐treatment. Scale bar: 5µm.
  6. Quantification of the CRMP4‐DIC puncta number per primary motor neuron axon in each condition. We analyzed ~ 20 axons per condition from 3 independent chambers per group (One‐way ANOVA, Tukey's multiple comparisons test, n = 3, data presented as mean ± SE, **P = 0.01, *P = 0.04).
  7. Representative images of proximity ligation assay for CRMP4 and dynein in healthy and C9orf72 human‐derived proximal axons post peptides treatment, Sema3A treatment, Sema3A + peptides treatment, or untreated controls. Scale bar: 5 µm.
  8. Quantification of the CRMP4‐DIC puncta number per axon in healthy or C9orf72 human‐derived MN proximal axons after Sema3A treatment, Sema3A + peptides treatment, or untreated controls. Data collected from 3 independent chambers in each condition. Total of 37 healthy untreated proximal axons, 61 healthy proximal axons with peptides treatment, 59 healthy proximal axons with Sema3A treatment and 52 healthy proximal axons with Sema3A + peptides treatment. 67 C9orf72 untreated proximal axons, 63 C9orf72 proximal axons with peptides treatment, 41 C9orf72 proximal axons with Sema3A treatment, and 50 C9orf72 proximal axons with Sema3A + peptides treatment monitored. Data presented as mean ± SE. One‐way ANOVA, Tukey's multiple comparisons test, n = 3, ****P < 0.0001.

Source data are available online for this figure.