Figure 3

PKCλ and PKMζ protein localize to central neurons in the dorsal horn and are increased by BDNF treatment. (A) Slices were prepared from mice and stained with an atypical PKC (aPKC) antibody (red) and the neuronal marker (found in soma and post-synaptic sites) SAP-102 (green). The top panel shows that aPKC immunoreactivity is localized primarily to the spinal dorsal horn. Lower panels (from inset) show that aPKC immunoreactivity is localized almost exclusively to neurons in the dorsal horn. (B) qPCR was used to assess mRNA expression of aPKC isoforms in spinal cord and kidney (N = 3). Spinal SNSs were prepared and mRNA or protein was isolated. Spinal SNSs were enriched for GluN1 mRNA and were βIII tubulin poor, they were likewise enriched in PSD95 protein as shown by Western blotting compared to equal protein concentration of whole spinal cord homogenate (input). C) SNSs were isolated from mouse lumbar spinal cord and exposed to increasing concentrations of BDNF for 15 min. BDNF dose-dependently increased mTOR, AKT and p70S6 kinase (p70) phosphorylation when standardized to total protein levels. BDNF also increased total levels of PKCλ PKMζ and CaMKIIα when compared to loading control βIII tubulin (N = 6). * p < 0.05, *** p < 0.001, one way ANOVA with Bonferroni post hoc test compared to Veh.