Figure 2. Specific N-terminal mutant htt fragments are enriched in brain mitochondria from HD knock-in mice. A, Forebrains from wild-type (7Q/7Q) or HD (150Q/7Q) knock-in mice were fractioned to purify mitochondria on a Percoll-based gradient. Whole-cell (Whole), cytosolic (Cyto), or mitochondrial (Mito) fractions (20 μg/each) were immunoblotted for various subcellular markers to confirm the purity of the isolated mitochondrial fraction. All mitochondrial proteins (30 and 70 kDa SDH, MnSOD, and AAT) showed significant enrichment in mitochondrial fractions. The blots were also probed with antibodies for plasma membrane (Na/K-ATPase), cytosolic (α-tubulin, GAPDH, G6PD, and NeuN), nuclear (NeuN), ER (BiP), endosomal (EEA1), or synaptosomal (synaptophysin) proteins. B, Subsequent blotting for mutant htt and full-length htt demonstrated a preferential association of N-terminal mutant-htt fragments with brain mitochondria. Note that the 1C2 antibody only reacts with mutant htt, whereas the 2166 antibody detects the full-length forms of both wild-type and mutant htt (bottom). C, Comparison of the sizes of N-terminal htt fragments in mitochondria isolated from HD knock-in mice with mutant htt of known lengths.