Detection of anti-PM/Scl antibodies in immuoprecipitation analysis. TnT-IPP: immunoprecipitation of biotinylated recombinant PM/Scl-100 and PM/Scl-75. Recombinant proteins were subjected to 4% to 20% SDS-PAGE and analyzed by immunoblotting with streptavidin-alkaline phosphatase and substrate. In., the input was half the dose for immunoprecipitation. Lanes A to K correspond to the anti-PM-Scl-100 and/or −75-positive patients shown in Figure 1. Lane N: healthy control serum. HeLa-IPP: immunoprecipitation analysis using radiolabeled HeLa cell extracts. Lanes A to L correspond to the patients shown in Figure 1 and Table 1. Lanes A to K correspond to anti-PM-Scl-100 and/or −75-positive patients shown in Figure 1. Lane M: [Methyl-14C] methylated protein MW markers (PerkinElmer Japan, Yokohama, Japan). Lane L: anti-U1-RNP-positive serum with equivocal titers for both antibodies in ELISA. Lanes 1 to 4 show the reference sera; lane 1, anti-PM/Scl-positive serum; lane 2, anti-MDA5-positive serum; lane 3, anti-TIF1-γ-positive serum; lane 4, anti-Mi-2-positive serum. Arrow and arrowhead corrspond to the PM/Scl-100 and PM/Scl-75 antigens, respectively. IPP, immunoprecipitation; TnT, in vitro translation and transcription product.