Fig. 4: Axotomy causes DLK-dependent phosphorylation of DRP1.

a Schematic representation of i3Neuron center plating and axotomy for protein harvesting after injury. b Representative western blots of WT and DLK KO neuron cell bodies untreated (UT), 2 and 4 h post axotomy. Immunoblot for p-S616 DRP1, total DRP1 and loading control ß actin. c Quantification of pDRP1 (S616)/ total DRP1 levels in WT and DLK KO neuron cell bodies untreated (UT), 2 and 4 h post axotomy. Results normalized to UT. N = 3 independent differentiations. One-way ANOVA. No significant changes observed. d Representative Western blots of WT and DLK KO neuron axons untreated (UT), 2 and 4 h post axotomy. Immunoblot for p-S616 DRP1, total DRP1 and loading control ß actin. e Quantification of pDRP1 (S616)/ total DRP1 levels in WT and DLK KO neuron axons 0, 2 and 4 h post axotomy. Results normalized to UT. N = 5 independent differentiations. Two-way ANOVA. Bonferroni correction (p = 0.0058 **). f Representative images of WT and DLK KO axons stained for ßIII tubulin (red) and pDRP1 S616 (white) untreated (UT), 30 min and 1 h post axotomy. Scalebar 25 µm. g Quantification of pDRP1 S616 fluorescence in WT and DLK KO neuron axons after axotomy. Results normalized to untreated axons. N = 3 independent differentiations. Two-way ANOVA, Bonferroni correction (UT vs 30 min p = 0.0156*, UT vs 1 h p = 0.0185*). h Schematic representation of optic nerve crush injury model. Bracket and * indicate the proximal portion of the injured nerve harvested for Western blotting. i Representative Western blots of proximal portion of ipsilateral (I) injured and contralateral (C) optic nerves 24 h after optic nerve crush. Immunoblot for p-S616 DRP1, total DRP1 and loading control ß actin. j Quantification of pDRP1 (S616)/ total DRP1 levels after optic nerve crush. Results normalized to contralateral side (C). N = 4 samples. Unpaired, two-tailed, t test (p = 0.0346 *). k Quantification of total DRP1/ ß actin levels after optic nerve crush. Results normalized to contralateral side (C). N = 4 samples. Unpaired, two-tailed t test. not significant, ns. All graphical representations show mean ± SEM.