Identification of the likely epitope for mEM48. (A) Comparison of N-terminal sequences of human and mouse htt and the antigen 2Q-htt. Two amino acid (VA) residues (italic type and underlined) are located after the polyP tract in human htt and 2Q-htt but are not present in mouse htt. Sequences in bold type were used for synthetic peptides. (B) Western blotting of cerebral cortical tissues from an HD YAC128 mouse, which expresses full-length mutant human htt, and from homozygous (Hom) or heterozygous (Het) HD 150Q knock-in (KI) mice, which express full-length mutant mouse htt. Note that the 1C2 antibody recognizes mouse and human mutant htt, whereas mEM48 only recognizes human mutant htt. (C) Htt sequences shown in A were used to synthesize N-terminal (N), human, mouse, or C-terminal (C) peptides. These peptides were applied to the dot blot at different concentrations (1–100 μM), and the blot was subsequently probed with mEM48. Only the human peptide reacts with mEM48. (D) PC12 cell lysates containing transfected GFP-htt (1–208 aa with 130Q) were immunoprecipitated by mEM48 in the presence of the human or mouse peptide (10 μm). The blot was probed with rabbit EM48. Only the human peptide blocks the immunoprecipitation. Control, immunoprecipitation without peptides. (E) Immunoprecipitation of scFv-EM48-HA in transfected PC12 cells by mouse anti-HA. The blot was probed with mEM48 for the coprecipitated GFP-htt-130Q (top, Htt) and with anti-HA for scFv-EM48 (bottom). The human but not the mouse peptide markedly inhibited the coprecipitation of htt.