Figure 1. hsc70-4 mRNA is a translation target of TDP-43^G298S and Hsc70-4 protein expression is reduced at synaptic terminals of TDP-43 expressing animals.

(A) Immunoprecipitation (IP) of TDP-43 expressed in motor neurons. Genotypes and antibodies used for IP indicated on top. Antibodies used for western blot (WB) indicated on right. (B) qPCR for hsc70-4 mRNA in TDP-43 complexes. (C) qPCR for hsc70-4 mRNA in soluble and insoluble TDP-43 complexes. (D–F) qPCR for hsc70-4 mRNA in input (D), RNP (E), and polysomes (F) from flies expressing TDP-43^WT and TDP-43^G298S in motor neurons compared to controls. (G) WB for Hsc70-4 levels in whole larvae expressing TDP-43 in motor neurons. Genotypes indicated on bottom. Actin was used as loading control. (H) Quantification of Hsc70-4 protein levels from WBs represented as a ratio to w¹¹¹⁸ controls. (I) qPCR for hsc70-4 mRNA in whole larvae of animals expressing TDP-43^WT or TDP-43^G298S in motor neurons versus controls. (J) WB for Hsc70-4 levels in VNCs of TDP-43 expressing larvae. Genotypes indicated on bottom. Actin was used as loading control. (K) Quantification of Hsc70-4 protein levels from WBs represented as a ratio to w¹¹¹⁸ controls. (L) qPCR for hsc70-4 mRNA in VNCs of animals expressing TDP-43^WT or TDP-43^G298S in motor neurons versus controls. (M–O) Single confocal sections of synaptic boutons in NMJ preparations immunostained for Hsc70-4 and the neuronal membrane marker Hrp from larvae expressing TDP-43^WT (N), and TDP-43^G298S (O) compared to w¹¹¹⁸ controls (M). Antibodies as indicated on left. (P) Quantification of Hsc70-4 intensity in synaptic boutons normalized to bouton area. Note p<0.001 for TDP-43^WT vs TDP-43^G298S. (Q) qPCR for hsc70-4 mRNA in NMJ preparations of animals expressing TDP-43^WT or TDP-43^G298S in motor neurons versus controls. (R) Quantification of Hsc70-4 intensity in muscle normalized to muscle area. Scale bars (M–O) 5 μm, 1 μm.