Figure 8.
Figure 8. ScFV-EM48 promotes the degradation of mutant htt. (A) Western blot of total lysates (T) and cytosolic (C), synaptosomal (S), and nuclear (N) fractions of striatal tissues from N171-82Q mice that had been injected with adenoviral vector (control) or scFv-EM48. Oligomeric mutant htt is present in the stacking gel (bracket). Arrows indicate transgenic htt and its cleaved product and arrowheads indicate additional degraded products. The same samples were probed with rabbit EM48 to htt and antibodies to the synaptic protein syntaxin, the nuclear protein TBP, and the cytoplasmic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The ratios (mean ± SEM, n = 3) of oligomeric or soluble htt to syntaxin were obtained using densitometry and are presented beneath the blots. *, P < 0.05. (B) Total lysates (T), cytoplasmic soluble (S), and pellet (P) fractions of PC12 cells coinfected by adenoviral GFP-htt-130Q and adenoviral vector (control) or scFv-EM48 were analyzed by Western blotting with mEM48 and antibodies to HA for scFV-EM48 or tubulin. Arrowheads indicate degraded htt products. (C) Pulse chase of mutant htt (GFP-htt-130Q) in adenoviral vector (control)– or scFv-EM48–infected PC12 cells (top). Arrowhead indicates a nonspecific band. Quantification of the percentage of remaining 35S-labeled htt signal at different chase times (bottom, n = 3). *, P < 0.05. (D) The pellet fractions described in B were analyzed by Western blotting with anti-ubiquitin (left) or subjected to EM48 immunoprecipitation (right). The precipitates were analyzed by Western blotting with anti-ubiqutin. The brackets indicate the increased amount of ubiquitinated products in the presence of scFv-EM48. Precipitated IgG is also indicated. (E) Immunofluorescent staining of PC12 cells, which express GFP-htt-130Q alone or with scFV-EM48, with rabbit anti-ubiquitin and mEM48 or mouse anti-HA. In the absence of scFv-EM48, mutant htt forms aggregates (arrows) that localize to the cell body and neurites (top). In the presence of scFV-EM48, htt aggregates (arrows) remain in the cell body and can be labeled by antibodies to ubiquitin (middle) or to the HA epitope in scFv-EM48 (bottom). Bars, 8 μm.

ScFV-EM48 promotes the degradation of mutant htt. (A) Western blot of total lysates (T) and cytosolic (C), synaptosomal (S), and nuclear (N) fractions of striatal tissues from N171-82Q mice that had been injected with adenoviral vector (control) or scFv-EM48. Oligomeric mutant htt is present in the stacking gel (bracket). Arrows indicate transgenic htt and its cleaved product and arrowheads indicate additional degraded products. The same samples were probed with rabbit EM48 to htt and antibodies to the synaptic protein syntaxin, the nuclear protein TBP, and the cytoplasmic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The ratios (mean ± SEM, n = 3) of oligomeric or soluble htt to syntaxin were obtained using densitometry and are presented beneath the blots. *, P < 0.05. (B) Total lysates (T), cytoplasmic soluble (S), and pellet (P) fractions of PC12 cells coinfected by adenoviral GFP-htt-130Q and adenoviral vector (control) or scFv-EM48 were analyzed by Western blotting with mEM48 and antibodies to HA for scFV-EM48 or tubulin. Arrowheads indicate degraded htt products. (C) Pulse chase of mutant htt (GFP-htt-130Q) in adenoviral vector (control)– or scFv-EM48–infected PC12 cells (top). Arrowhead indicates a nonspecific band. Quantification of the percentage of remaining 35S-labeled htt signal at different chase times (bottom, n = 3). *, P < 0.05. (D) The pellet fractions described in B were analyzed by Western blotting with anti-ubiquitin (left) or subjected to EM48 immunoprecipitation (right). The precipitates were analyzed by Western blotting with anti-ubiqutin. The brackets indicate the increased amount of ubiquitinated products in the presence of scFv-EM48. Precipitated IgG is also indicated. (E) Immunofluorescent staining of PC12 cells, which express GFP-htt-130Q alone or with scFV-EM48, with rabbit anti-ubiquitin and mEM48 or mouse anti-HA. In the absence of scFv-EM48, mutant htt forms aggregates (arrows) that localize to the cell body and neurites (top). In the presence of scFV-EM48, htt aggregates (arrows) remain in the cell body and can be labeled by antibodies to ubiquitin (middle) or to the HA epitope in scFv-EM48 (bottom). Bars, 8 μm.

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