DAR2B in CCI-mice MedChemExpress PP 242 treated with compound 30 compared to vehicle group. No significant changes were observed in the total levels of NMDAR2B in any PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19770275 group. Variations in the levels of tyrosine phosphorylation of NMDAR2B could be related to a stable surface expression through palmitoylation. To know whether the decrease of phospho-NMDAR2B observed in CCI-mice treated with compound 30 was associated with a reduction in the surface membrane expression, we analyzed by 8 / 15 A Novel EGCG Derivative for Reducing Neuropathic Pain after CCI Fig 3. Spinal cord of CCI-mice treated with EGCG and compound 30 at 14 dpi showed decreased levels of inflammatory cytokines Total RNA of the dorsal horn of spinal 
cord of control mice and CCI-mice treated with vehicle and 50 mg/Kg of EGCG, compound 23 and compound 30 was isolated at 14 and 56 days post injury as indicated in materials and methods. Quantitative RT-PCR analysis of TNF-, IL-1 and IL-6 was performed. Data were expressed as fold change normalized to 18S and represent the mean SEM. p<0.05 and p<0.001 compared to vehicle-treated CCImice using a one-way ANOVA with Bonferroni's post-hoc test. Protein extracts from the dorsal horn of the spinal cord of control mice and CCI-mice treated with vehicle and 50 mg/Kg of EGCG, compound 23 and compound 30 at 14 and 56 days post injury were subjected to western blot to study the TNF-, IL-1 and IL-6 protein levels. Data were expressed as a percentage with respect to shammice. Results are the mean SEM and represent the ratio between each protein and actin levels, obtained by densitometric analysis of western blot. Data were analyzed by one-way ANOVA with Bonferroni's post-hoc test. p<0.001 compared to sham PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19769788 mice and +++p<0.001 compared to vehicle-treated CCI-mice. Representative immuno-blots are presented. doi:10.1371/journal.pone.0123122.g003 Western blot the levels of NMDAR2B in a synaptic plasma membrane fraction of the dorsal horn of the spinal cord at 56 dpi. We strongly detected a specific decrease in the protein levels of NMDAR2B in the SPM of CCI-mice treated with compound 30. The administration of EGCG and compound 23 did not result in changes in NMDAR2B levels in the SPM fractions. These results suggest that chronic prevention of CCI-induced 9 / 15 A Novel EGCG Derivative for Reducing Neuropathic Pain after CCI Fig 4. Reduced nuclear levels of NF-B in the spinal cord CCI-mice administrated with EGCG and compound 30 at 14 dpi. Nuclear protein extracts from the dorsal horn of the spinal cord of control mice and CCI-mice treated with vehicle and 50 mg/Kg of EGCG, compound 23 and compound 30 at 14 and 56 days post injury were subjected to western blot to study the NF-B protein levels. Data were expressed as a percentage with respect sham-mice. Results are the mean SEM and represent the ratio between each NF-B and NeuN levels, obtained by densitometric analysis of western blot. Data were analyzed by one-way ANOVA with Bonferroni's post-hoc test. p<0.001 compared to sham mice and +++p<0.001 compared to and vehicle-treated CCI-mice. Representative immuno-blots are presented. doi:10.1371/journal.pone.0123122.g004 Fig 5. Decreased levels of NMDAR2B in the synaptic plasma membrane of spinal cord of CCI-mice treated with compound 30 at 56 dpi. Protein extracts from the dorsal horn of the spinal cord of control mice and CCI-mice treated with vehicle and 50 mg/Kg of EGCG, compound 23 and compound 30 at 14 and 56 days post injury were subjected to western blot to study
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