Figure 6

Mutant VAPB inclusions are ubiquitin-positive and enriched in ERAD components. A-C) Representative confocal images of the distribution of ubiquitinated epitopes labeled with the FK2 antibody in motor neurons of mutant VAPB line VM1 with (A) or without (B) inclusions, and non-transgenic littermates (C). Note high level of FK2-immunoreactivity in inclusions (A) and diffuse increase as compared to non-transgenic motor neurons in VM1 motor neurons without inclusions (B). D) Bar graph of FK2-immunofluoresencesignal in non-transgenic versus VM1 and VM3 motor neurons without inclusions. Values are means ± SE and are from lumbar L4 segments from 4 non-transgenic, 2 VM1 and 2 VM3 mice embedded in a single gelatin block (see methods). E-I) Confocal image of mutant VAPB (line VM1, E-G) or non-transgenic (H, I) spinal motor neurons double-labeled for VAPB or HA and ERAD components, i.e. VCP/p97 (E, I), derlin-1 (F, H), and BAP31 (G). Note, enrichment of VCP (E) and BAP31 (G) staining in the mutant VAPB inclusions; derlin-1 although present does not show preferential enrichment is not selectively enriched in the mutant VAPB inclusions. Scale bars: 10 μm (A, H), 25 μm (E).