Figure 6
3B12A scFv-CMA induces HSP70 expression, which leads to the unfolding of TDP-43 aggregates. (a–d) Measurement of HSP70 expression in the presence of TDP-43 and 3B12A scFv. (a) Western blot analysis of endogenous HSP70 and HSC70 protein expression levels. (b,c) Quantified densitometric analysis of HSP70 protein ratio (b) and HSC70 and HSP70 protein ratio (designated as HSC70 and HSP70) obtained from immunoblots using an antibody recognising both HSC70 and HSP70 (N27F3-4) (c) Each data point was obtained by normalisation to actin. Differences were evaluated by one-way ANOVA (mean ± SD from three independent experiments; **p < 0.01 vs vector). (d) Quantitative real-time PCR analysis of HSP70 gene expression. Differences were evaluated by one-way ANOVA (mean ± SD from triplicates; ***p < 0.005, ****p < 0.001 vs vector). N.S. indicates not significant. (e) Confocal microscopic analysis of HEK293A cells expressing GFP-fused TDP-43. Endogenous HSP70 colocalised with both nuclear and cytoplasmic TDP-43 aggregates (arrow heads), whereas HSP70 did not colocalise with TDP-43WT. Scale bar = 20 µm. (f) Interaction of endogenous HSP70 and overexpressed TDP-43 species. HSP70 interacted with cytoplasmic aggregated TDP-43 predominantly in the presence of VH_VL-CMA. DCS indicates the C173S/C175S mutant. (g) Western blot analysis for detergent insolubility of TDP-43 in the presence of HSP70 or HSP90 overexpression in HEK293A. Cell lysates were separated into RIPA-soluble or -insoluble fractions after centrifugation for 20 min at 15,000 × g at 4 °C and subsequently eluted in 2% SDS sampling buffer for 5 min at 95 °C. DCS represents the C173S/C175S mutant. (h) Quantified densitometric analysis of (g) Each data point was obtained by normalisation to actin. The insolubility ratio is normalised insoluble TDP-43 to normalised soluble TDP-43. Differences were evaluated by one-way ANOVA (mean ± SD from three independent experiments; *p < 0.05 vs vector).
