IB, Immunoblotting

IB, Immunoblotting. by attenuating TNIK/GluR1 phosphorylation-dependent subcellular GluR1 redistribution. In contrast, intrathecal administration of BC-1215 (analysis), two-way ANOVA (using Tukey’s assessments for analysis), or paired Student’s test when appropriate. Significance was set at 0.05. In experiments exploring the effects of treatments on SNL-induced protein expression (WB), conversation (IP), and location [immunohistochemistry (IHC)], one-way ANOVA was first used to compare the statistical difference among groups. In case the difference was significant, Tukey’s test was used to examine the difference between sham-operated and SNL animals. If the value of SNL is usually statistically different from the sham-operated animals, then Tukey’s test was used to compare SNL animals with SNL animals that received numerous treatments. Results SNL induces pain-associated dorsal horn TNIK expression As the first to address whether Atglistatin spinal TNIK plays a role in neuropathic pain, TNIK expression in the dorsal horn (L4CL5) after SNL was examined. SNL increased significantly the large quantity of TNIK in the ipsilateral, but not the contralateral, dorsal horn at days 3, 7, 14, and 21 after surgery (0.42 0.03, 0.58 0.05, 0.52 0.03, and 0.48 0.05, respectively; = 6; Fig. 1= 7; Fig. 1= 7; Fig. 1= 0.002; time, 0.001; and treatment time, 0.001. ** 0.01 Atglistatin versus Sham IPSI; ## 0.01 versus SNL day 1. = 6. IB, Immunoblotting. 0.001; time, = 0.016; and treatment time, 0.001. ** 0.01 versus Sham IPSI; ## 0.01 versus day 1. = 7. Tukey’s test: 0.001. ** 0.01 versus Sham IPSI; ## 0.01 versus SNL CONTRA. = 7. Level bar, 50 m. Knockdown of TNIK expression alleviates SNL-induced allodynia To further confirm the role of TNIK in neuropathic pain hypersensitivity, we examined whether the lack of spinal TNIK affects the development of SNL-induced allodynia. An intrathecal injection of TNIK mRNA-targeting siRNA (1, Atglistatin 3, and 5 g, 10 l, once daily for Atglistatin 4 d), but not missense siRNA (5 g, 10 l) or polyethylenimine (a transfection reagent, 10 l), decreased the large quantity of TNIK in dorsal horn samples (0.35 0.06, 0.15 0.03, and 0.11 0.02, respectively; = 6; Fig. 2= 7; Fig. 2Tukey’s test: 0.001. ** 0.01 versus Naive. = 6. IB, Immunoblotting; it, implantation of an intrathecal catheter. = 0.698; time, = 0.823; treatment time, = 0.865. = 7. = 0.981; time, = 0.683; treatment time, 0.999. = 7; In SNL animals, two-way ANOVA with repeated steps over time: treatment, 0.001; time, 0.001; treatment time, 0.001. ** 0.01 versus SNL. = 7. SNL consecutively provoked spinal GluR1 coupling with TNIK, phosphorylation, and trafficking TNIK, as a serine/threonine kinase and a scaffold domain name, has been shown recently to regulate cell-surface GluR1CAMPAR trafficking (Hussain et al., 2010). Our laboratory has shown previously that both serine/threonine kinases (Peng et al., 2012b) and postsynaptic scaffolding proteins (Peng et al., 2011) drive the phosphorylation-dependent subcellular trafficking of spinal GluR1CAMPARs to underlie the pain-associated central sensitization. Thus, we hypothesized that SNL-enhanced TNIK expression provokes spinal GluR1 phosphorylation and subsequent trafficking. We found that SNL significantly increased the large quantity of GluR1, but not tGluR1, in the ipsilateral dorsal horn samples at days 3 and Atglistatin 7 after the operation (0.58 0.05 and 0.62 0.04, respectively; = 6; Fig. 3= 6), suggesting that this phosphorylation-dependent subcellular trafficking of spinal GluR1CAMPARs plays a crucial role in SNL-induced neuropathic pain. Strikingly, we found that daily administration of TNIK mRNA-targeting siRNA (5 g, 10 EFNB2 l) significantly and quantitatively reversed SNL-enhanced abundances of pGluR1 (Fig. 3= 6).