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. 2017 Oct 13;27(12):1441–1465. doi: 10.1038/cr.2017.113

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Copyright © 2017 The Author(s) 2017

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Mapk11 and Hipk3 regulate Htt levels via their kinase activities. (A) Representative western blots and quantifications of Htt levels in the HD (STHdh^Q7/Q111) cells or WT (STHdh^Q7/Q7) cells transfected with the indicated siRNAs. (B) Representative western blots and quantifications of Htt levels in the HD (STHdh^Q7/Q111) transfected with dominant negative MAPK11 cDNA (DN-MAPK11) versus MAPK11 cDNA (MAPK11) plasmids. (C) Representative western blots and quantifications of Htt levels in the HD (STHdh^Q7/Q111) transfected with indicated siRNAs targeting Mapk11 and cDNA plasmids expressing MAPK11 or DN-MAPK11. (D) In primary cultured striatal neurons from Hdh^Q7/Q140 mice transfected with the scramble control siRNA or the Mapk11 siRNA (Mapk11_si2), the dose-dependent curves of the Htt levels upon treatment of the p38 Mapk inhibitor SB202190 were measured by HTRF using the 2B7/2166 antibody pair. Data plotted as mean and SEM, n = 6. Curves were fitted by the Sigmoidal dose-dependent curve. (E) Representative western blots and quantifications showing that over-expression of the wild-type Hipk3 cDNA (HIPK3) increases Htt levels in the STHdh^Q7/Q111 cells, while the kinase-dead Hipk3 (K226M and D322N) has no such effects. (F) Left, Representative western blots showing that treatment with the Hipk3 inhibitor AST487 reduces Htt levels in STHdh^Q7/Q111 cells at 10 μM. Right, in the STHdh^Q7/Q111 cells transfected with the scramble control siRNA or the Hipk3 siRNA (Hipk3_si1), the dose-dependent curves of the Htt levels upon treatment of AST487 were measured by HTRF using the 2B7/2166 antibody pair. Transfection of the Hipk3 siRNA abolished the Htt lowering effect of AST487. Data plotted as mean and SEM, n = 10. Curves were fitted by the Sigmoidal dose-dependent curve. (G) Similar to (F), but in immortalized HD patient fibroblasts (Q68). In the HTRF experiments in the right panel, HD Q68 fibroblasts were transfected with the scramble control siRNA or the HIPK3 siRNA (HIPK3_si1), and the mHTT levels were measured by HTRF using the 2B7/MW1 antibody pair. Transfection of the HIPK3 siRNA abolished the effect of AST487. Data plotted as mean and SEM, n = 10. Curves were fitted by the Sigmoidal dose-dependent curve. (H) In the HD patient iPSC-derived neurons (Q47), mHTT levels upon treatment of AST487 were measured by HTRF using the 2B7/MW1 antibody pair. Data plotted as mean and SEM, n = 12. The statistical analysis was performed by one-way ANOVA and Dunnett's post hoc tests. ^**P < 0.01.