A mutant variant of CRMP1 (D408V) shows impaired activity in cell-free HTT aggregation assays. (A) The proteins His-CRMP1 and His-CRMP1-D408V were produced in E. coli and purified to >90% homogeneity by affinity chromatography. Protein expression and purity was confirmed by Coomassie staining of SDS gels or immunoblotting (IB). (B) Analysis of spontaneous HTTQ49 aggregation in the presence of modulator proteins in cell-free assays by filter retardation assay. SDS-insoluble HTTQ49 protein aggregates were detected on filter membranes using the anti-HTT antibody CAG53b. Representative results from three independent experiments are shown. (C) Analysis of spontaneous HTTQ49 aggregation in the presence of modulator proteins by dot-blot assays. The conversion of soluble HTTQ49 into insoluble protein aggregates was monitored using the antibody 3B5H10. Representative results from three independent experiments are shown.
