Generation and expression of scFV-EM48. (A) Structure of the intrabody scFv-EM48. The intrabody (251 aa) consists of variable region heavy and light (V_H and V_L) chains of the scFv for mEM48, which are linked by a Gly-Ser domain and tagged with the HA epitope. Amino acid numbers are indicated. (B) Expression of scFv-EM48-HA in cultured HEK293 cells. scFv-EM48-HA was either transfected alone or cotransfected with mutant htt (1–208 aa) containing 130Q. Cells were labeled with mouse anti-HA for scFv-EM48 and rabbit EM48 for htt. Note that scFv-EM48 is colocalized with mutant htt (htt-130Q) in cytoplasmic aggregates. (C) Coexpression of scFv-EM48 and mutant htt in cultured rat cortical neurons (arrows) also shows the colocalization of scFv-EM48 with mutant htt. In B and C, cells were immunostained with mouse anti-HA (12CA5) and rabbit EM48. Bars, 10 μm. (D) Western blot analysis of immunoprecipitates from transfected HEK293 cells that express scFv-EM48-HA and exon1 htt with 130Q or 20Q. scFv-EM48 (scFv) was precipitated by the antibody (12CA5) to the HA epitope (bottom). The input and precipitates (IP) were then probed with rabbit EM48 (top). The arrow indicates soluble htt-20Q and the arrowhead indicates htt-130Q. The bracket indicates the stacking gel in which aggregated htt is seen. (E) Densitometry of the ratio of precipitated htt to input from a representative experiment. Similar results were seen in two separate experiments.