FIGURE 2.

Anti-Jo-1 patient serum reacts mainly with the N-terminal WHEP domain and C-terminal ABD of human HisRS, and recombinant HisRS SVs are secreted from HEK293T cells and C2C12 myoblasts. A, illustration of depletion ELISAs. Details are provided under “Experimental Procedures.” B, reactivity of anti-Jo-1 Ab-positive patient serum against different HisRS recombinant proteins. Notably, apart from HisRS, anti-Jo-1 Ab-positive patient serum reacted mostly with HisRS^WHEP and HisRSΔCD, with a significantly higher reactivity than with the recombinant CD or ABD. C, the two most reactive domains are far apart on the three-dimensional structure of HisRS (Protein Data Bank code 4G84). The C-terminal ABD (shown in green) and N-terminal WHEP domain (shown in red) are highlighted in the structure of HisRS. The N-terminal WHEP domain is not resolved in the structure of HisRS, but is resolved in that of HisRSΔCD (Protein Data Bank code 2LW7). D, the structures of HisRSΔCD and HisRS^WHEP show that the two HisRS SVs contain the major anti-Jo-1 epitopes. E and F, recombinant HisRS^WHEP, HisRSΔCD, and HisRS proteins (with a C-terminal Myc tag) were transiently expressed in HEK293T cells (E), and recombinant HisRSΔCD and HisRS proteins were transiently expressed in C2C12 myoblasts (F). Expressed proteins were detected in the TCLs with anti-Myc mAb. LDHB in the TCLs served as a loading control. The media were immunoprecipitated by anti-Myc polyclonal Ab and detected with anti-Myc mAb. Notably, all HisRS proteins were detected in the media. The bar graphs show that the LDH activities of all samples were below the detection limit of the assay (indicated by the red line), suggesting little cell damage. The results shown are representative of three separately conducted experiments.