fig4

Figure 4. VAPB P56S mutation destabilizes the mutant protein in a proteasome-dependent way. (A-E) Costaining of HSP70 (A); ubiquitin (B); BAP31 (C); VCP (D); or SQSTM1 (E) with VAPB and CTIP2 (red) in the CSMNs of WT and P56S KI mice at 2 months of age. The colocalization of HSP70 (A); ubiquitin (B); BAP31 (C); VCP (D); or SQSTM1 (E) with VAPB inclusions in P56S KI mice was marked with arrows. (F-I) Western blotting of VAPB in DIV28 WT and P56S KI cortical neurons after 48-hour treatment with 1 μg/mL CHX (F) or 0.1 μm MG132 (H). The VAPB decrease after CHX treatment (G) and the VAPB increase after MG132 treatment (I) were quantified (n = 4). (J and K) Immunostaining of VAPB, MAP2, and ankyrin G (purple) in DIV28 WT and P56S KI cortical neurons after indicated treatment for 48 h (n = 4). Scale bar: 20 μm in (A-E, and J). Unpaired t test: ^****P < 0.0001 in (G); ^****P < 0.0001 in (I); One-way ANOVA: ^****P < 0.0001 [P56S KI (vehicle) vs. WT], P < 0.0001 [P56S KI (CHX) vs. WT], P < 0.0001 [P56S KI (MG132) vs. WT], ^**P = 0.0016473382 [P56S KI (CHX) vs. P56S KI (vehicle)], ^***P = 0.0007940139 [P56S KI (CHX) vs. P56S KI (vehicle)], and ^**P = 0.0016473382 [P56S KI (CHX) vs. P56S KI (MG132)] in (K). VAPB: Vesicle-associated membrane protein-associated protein B; HSP70: heat shock protein 70; BAP31: B-cell receptor-associated protein 31; VCP: valosin-containing protein; SQSTM1: sequestosome 1; CTIP2: COUP-TF-interacting protein 2; ANOVA: analysis of variance; WT: wild-type.