Fig. 5: P56S-VAPB and altered distribution of other fALS-associated RBP.

a–c Co-labelling of HeLa cells expressing wt-VAPB and P56S-VAPB with antibodies against TDP43 (a) with FUS (b) and with Matrin 3 (c). Note that the cytoplasmic aggregation (arrowheads) and reduced nuclear immunoreactivity (arrows) of these RBPs in P56S-VAPB expressing cells. Scale bar: 10 µm. d Immunoblot analysis of the subcellular fractions obtained from HeLa cell expressing wt-VAPB and P56S-VAPB. Note the increased levels of TDP-43, FUS and of Matrin 3 (red arrowheads) in cytoplasmic extract (CE). ME membrane extract, NE nuclear extract, CB chromatin bound, PE cytoskeleton. e DAB immunohistochemistry using antibodies against pTDP-43 and Matrin 3, showing altered nuclear immunoreactivity (black arrows) and diffuse albeit increased cytoplasmic immunoreactivity (white arrows) in lumbar spinal cord α-MNs of P56S-VAPB tg mice. Scale bars: 15 µm. f Double immunofluorescence labelling for HA and for FUS antibodies showing reduced labelling of FUS (arrow) in lumbar spinal cord α-MNs of P56S-VAPB tg mice. Scale bars: 15 µm. g Prominent and globular pTDP-43 immunoreactivity (white arrows- upper panel; DAB, lower panel; immunofluorescence) of grouped angular or flattened, atrophic, denervated muscle fibres in the ALS8 biopsy. Scale bars: 80 µm. h Co-localisation of globular and aggregated pTDP43 with VAPB aggregates (white arrows) in the same biopsy. Scale bars: 60 µm. i DAB immunohistochemistry of Matrin 3, showing increased immunoreactivity of the myonuclei (arrows) and mildly diffuse saromeric accumulations. Note the basal myonuclei immunoreactivity of Matrin 3 in non-atrophic normal-sized fibres (left panel). Co-immunolabelling (lower panel) with pTDP-43 antibody showed no co-localisation between Matrin 3 accumulations and pTDP-43 aggregates. Scale bars: 80 µm.