Fig. 6: Extracellular Pgk1 interacts with eno2 on the neural membrane to reduce p-Cofilin, enhancing neurite outgrowth of motor neurons in zebrafish embryos.

a, b Phenotypes of motor neurons observed in the embryos from transgenic line Tg(mnx1:GFP): a normal axonal phenotype neurons; b branched axonal phenotype neurons (indicated by white arrows). Scale bar: 50 μm. c After embryos were incubated, either with (+) or without (−) zebrafish Pgk1 for 24 h, they were injected with or without three isoforms of eno-mRNAs. The percentage of embryos displaying branched motor neuron phenotype among the total number (n) of examined embryos in each group was calculated. Data were averaged from three independent experiments. Data were averaged from three independent experiments and presented as mean ± SD (n = 3). One-way ANOVA, followed by Tukey’s multiple comparison test, was used to perform statistical analysis (**P < 0.005 and NS, not significant, P > 0.05). d Western blot analysis. Zebrafish embryos from transgenic line Tg(mnx1:GFP) were incubated with (+) or without (−) zebrafish Pgk1 for 24 h and injected with or without three isoforms of eno-mRNAs. Non-injected embryos served as control, followed by collecting total protein lysate from Tg(mnx1:GFP) and then analyzing the levels of phosphorylated Cofilin at S3 (p-Cofilin) and total Cofilin. The α-tubulin served as an internal control. The change (in fold) of relative intensity of each examined protein against internal marker α-tubulin compared to that of the zPgk1-control group relative to each internal control set as 1 was presented below each lane. Data were averaged from three independent experiments. e Statistical analysis. The change (in fold) of relative intensity of p-Cofilin against internal marker tubulin compared to that of the control group which was set as 1. Data were averaged from three independent experiments and presented as mean ± SD (n = 3). One-way ANOVA, followed by Tukey’s multiple comparison test, was used to perform statistical analysis (**P < 0.005 and NS, not significant, P > 0.05). The same protein extracts from each experiment were loaded onto separate western blots and probed for individual proteins, in parallel.