Journal of Neurodegenerative Diseases / 2016 / Article / Fig 3

Research Article

An Intrabody Drug (rAAV6-INT41) Reduces the Binding of N-Terminal Huntingtin Fragment(s) to DNA to Basal Levels in PC12 Cells and Delays Cognitive Loss in the R6/2 Animal Model

Figure 3

INT41 reduces binding of mHtt fragments to chromatin in PC12 cells: PC12 cells (nHtt Q23 and mHtt Q73) were transduced with rAAV6-INT41 (41) or mock-transduced (M) and then induced for Htt expression after 72 hours with Ponasterone A. Cells were harvested 8 days after induction and then subjected to subcellular fractionation as described (Section 2). 10 μg protein from each fraction was separated on a 3–8% Tris-Acetate gel, then transferred to nitrocellulose, and (a) immunoblotted with rabbit anti-N-terminal Htt antibody and secondary as described (Section 2). (b) Immunoblot with a mouse anti-CREB, anti-GADPH, or anti-Histone H3 as markers for subcellular fractionation. Molecular weights are in kilodaltons as indicated. (c) INT41 distribution into subcellular compartments. A blot from subcellular distribution was reprobed with affinity purified rabbit anti-INT41 to obtain intrabody distribution between cytoplasmic (C), membrane (M), nuclear soluble (NS), and nuclear chromatin (NC) fractions for both mock- (M) and rAAV6-INT41-transduced cells (41). (d) Schematic representation of Caspase 3 and Caspase 6 cleavage of Htt.