Fig. 5: Extracellular Pgk1 interacting with membrane protein Eno2, but neither Eno1 nor Eno3, caused the reduction of p-Cofilin, resulting in enhancing neurite outgrowth of motoneurons derived from neural cells. | Communications Biology

Fig. 5: Extracellular Pgk1 interacting with membrane protein Eno2, but neither Eno1 nor Eno3, caused the reduction of p-Cofilin, resulting in enhancing neurite outgrowth of motoneurons derived from neural cells.

From: Extracellular Pgk1 interacts neural membrane protein enolase-2 to improve the neurite outgrowth of motor neurons

Fig. 5

a Morphology of neurites derived from NSC34 neural cells was observed under microscopy. Five experimental groups were categorized: (1) cells treated with pCS2 transfection, (2) pCS2 transfection plus Pgk1 incubation (pCS2+Pgk1), (3) pCS2-Eno1 transfection plus Pgk1 incubation (pCS2-Eno1+Pgk1), (4) pCS2-Eno2 transfection plus Pgk1 incubation (pCS2-Eno2+Pgk1), and (5) pCS2-Eno3 transfection plus Pgk1 incubation (pCS2-Eno3+Pgk1). Scale bar, 100 μm. b Neurite Outgrowth Analysis Application Module of MetaMorph software was used to count neurite-bearing cells and measure neurite lengths. Cell body was marked in white, while neurite was marked in red. c, d Statistical analysis: c percentage of neurite-bearing cells among examined cells and d average neurite length of each group, as indicated. 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). e Western blot analysis. NSC34 cells incubated either in the presence (+) or absence (−) of Pgk1 and transfected either pCS2-Eno1-, -Eno2-, or -Eno3-Myc, followed by 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 α-tubulin of each group compared to that of the pCS2-control group relative to each internal control set as 1 was presented under each lane. Data were averaged from three independent experiments. f Statistical analysis. 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.

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