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. 2015 Dec 16;35(50):16545–16560. doi: 10.1523/JNEUROSCI.2301-15.2015

Figure 6.

Figure 6.

BC-1215 relieves allodynia by preventing ubiquitination-dependent spinal Fbxl2/TRAF2/TNIK/pGluR1 signaling. A, Representative WB and statistical analyses (normalized to GAPDH) demonstrating SNL decreased Fbxl2 but increased Fbxo3 abundance in the ipsilateral (IPSI) dorsal horn. One-way ANOVA, post hoc Tukey's test: Fbxl2, F(2,15) = 20.63, p < 0.001; Fbxo3, F(2,15) = 11.21, p = 0.001. **p < 0.01 versus Sham. ##p < 0.01 versus SNL day 1. n = 6. B, C, Intrathecal administration with BC-1215 (SNL 7D+BC-1215, 10, 30, and 100 nm, 10 μl), but not the vehicle solution (SNL 7D+Veh, 10 μl), increased the withdrawal threshold of the ipsilateral hindpaw. Nevertheless, both these treatments failed to affect the contralateral hindpaw at 3 h after injection (BC-1215 100 nm; von Frey test). Ipsilateral hindpaw, two-way ANOVA with repeated measures over time: treatment, F(4,30) = 46.98, p < 0.001; time, F(6,180) = 22.40, p < 0.001; treatment × time, F(24,180) = 6.460, p < 0.001. *p < 0.05, **p < 0.01 versus SNL 7D. n = 7. Contralateral hindpaw, one-way ANOVA, post hoc Tukey's test: F(2,18) = 0.031, p = 0.970. D, At day 7 after surgery, SNL increased Fbxl2 ubiquitination in the ipsilateral dorsal horn, which was reversed markedly by spinal injection of BC-1215, which increased TRAF2 ubiquitination. E, F, Without affecting the abundances of tGluR1 and Fbxo3, BC-1215 reversed the SNL-increased TRAF2, TNIK, pGluR1, and GluR1(m) expressions and the SNL-decreased Fbxl2 expression. One-way ANOVA, post hoc Tukey's test: TRAF2, F(3,20) = 48.08, p < 0.001; TNIK, F(3,20) = 18.75, p < 0.001; Fbxl2, F(3,20) = 43.52, p < 0.001; Fbxo3, F(3,20) = 16.80, p < 0.001; pGluR1, F(3,20) = 26.66, p < 0.001; tGluR1, F(3,20) = 0.260, p = 0.854; GluR1(m), F(3,20) = 28.80, p < 0.001. **p < 0.01 versus Sham 7D. ##p < 0.01 versus SNL 7D. n = 6. G, Administration with neither TNIK mRNA-targeting siRNA (SNL 7D+TNIK RNAi, 5 μg, 10 μl) nor missense siRNA (SNL 7D+TNIK MS, 5 μg, 10 μl) affected SNL-upregulated Fbxo3 and SNL-downregulated Fbxl2 expression. One-way ANOVA, post hoc Tukey's test: Fbxl2, F(3,20) = 23.94, p < 0.001; Fbxo3, F(3,20) = 4.992, p = 0.010. *p < 0.05, **p < 0.01 versus Sham 7D. n = 6. H, SNL-enhanced tGluR1-TRAF2/TNIK/pGluR1 precipitations were reduced by spinal injection of BC-1215.