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. 2015 Jul 14;34(17):2255–2271. doi: 10.15252/embj.201490808

graphic file with name embj0034-2255-f10ev.jpg

Intramolecular interactions between proteolytic huntingtin fragments

  1. Immunoprecipitations upon cleavage of FL-HTT-TEV-Q23 by the SNIPer-TEV performed in HEK293T cell. * indicates a non-specific band.
  2. Immunoprecipitations of HeLa cell extracts expressing mCherry-N-HTTQ100 constructs or mCherry and C-HTT587-3144-GFP or GFP using anti-mCherry antibody.
  3. Immunoblotting analysis of the expression level of N- and C-HTT alone or in combination with striatal cells. * indicates a non-specific band.
  4. Toxicity in striatal cells transfected with FL-HTT167/586TEV-Q23 and FL-HTT167/586TEV-Q100 and siRNA targeted against endogenous HTT (si-HTT). Graph summarising cell death ratio at 24 h of proteolysis-induced toxicity is shown as a ratio of the percentage of cell death obtained in SNIPer-TEV condition over pcDNA condition. Downregulation of HTT expression was assessed by immunoblotting (right). The bar graphs (mean ± SEM) display pooled data from two to four independent experiments. Total number of cells is as follows: FL-HTT167/586TEV-Q23 without si-HTT: 399 cells; with si-HTT: 230 cells; FL-HTT167/586TEV-Q100 without si-HTT: 430 cells; with si-HTT: 199 cells. Statistics were done by unpaired t-test. ns: non-significant.