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. 2013 Jun 27;8(6):e68055. doi: 10.1371/journal.pone.0068055

Figure 2. Hc-TeTx induces nSMase activity.

Figure 2

A) CGN were incubated at different times with 10 nM Hc-TeTx and SMase activity was measured at pH 7.5 using the Amplex Red Sphingomyelinase Assay Kit (Molecular Probes, UK). Alternatively, cells were pretreated for 1 h with 20 µM GW4869, a specific inhibitor for nSMase, previously to the Hc treatment. Each point represents the mean ± SD of three independent experiments. B) The graphic shows the increase in nSMase activity after 1 h of incubation with NGF 50 ng/mL, which is totally prevented by pretreatment with 20 µM GW4869. Shown are mean ± SD of three independent experiments. Values were analyzed by one-way ANOVA followed by Bonferroni post hoc test to compare SMase activity in Hc-treated cells respect to controls. ***P<0.0001. C) Cultured cortical neurons (CCN) were incubated at different times with 10 nM Hc-TeTx or with NGF 50 ng/mL and SMase activity was measured at pH 7.5 using the Amplex Red Sphingomyelinase Assay Kit (Molecular Probes, UK). Each point represents the mean ± SD of three independent experiments.