Mutant VAPB inclusions in primary cultured hippocampal neurons are enriched in ERAD components. A-E) Maximal projections of confocal stacks of primary cultured hippocampal neurons transfected with Myc-VAPB-P56S showing intense staining of ubiquitinated epitopes (FK2, A), derlin-1 (B), VCP (C, D) and BAP31 (E) in mutant VAPB inclusions. F, G) Co-expression of Myc-VAPB-P56S and RFP-BAP31 shows that mutant VAPB inclusions recruits a large portion of RFP-BAP31 (F), while in single RFP-BAP31 transfected neurons, RFP-BAP31 has a diffuse distribution (G). H, I) Hippocampal neurons transfected with GFP-tagged VAPB-P56S lacking the transmembrane domain (ΔTM) develop inclusions that are immunonegative for VCP (H) and Derlin-1 (I). J, K) Transfection of hippocampal neurons with VAPB-P56S-CD8TM with (K) or without (J) myc-VAPB-P56S (green) shows that VAPB-P56S with the transmembrane domain of CD8 (immunostained with anti-CD8 antibody) accumulates in the same inclusions as VAPB-P56S. Scale bar, 10 μm.