Immumofluorescent ANA and western blot with anti-RPA positive sera (a) Immunofluorescent ANA testing with anti-RPA-positive sera. HEp-2 cells were stained with mAb against RPA32 (i), RPA70 (ii), human autoimmune sera with anti-RPA (1:160 dilution, iii–vii), or normal control (viii). All anti-RPA-positive sera showed nuclear fine speckled/homogeneous staining, similar to the staining by anti-RPA32 or anti-RPA70 mAb. Some sera had an additional immunofluorescent pattern from the other coexisting specificities; mitochondria (vi) and centromere (vii). (b) Western blot analysis of anti-RPA antibodies. RPA was immunoprecipitated from K562 cell extract, fractionated by 12% SDS-PAGE, and transferred to a nitrocellulose filter. Strips of the filter were probed with mAbs against RPA (lanes a to d: a, RPA14; b, RPA32; c, RPA32; d, RPA70), anti-RPA immunoprecipitation-positive sera (lanes 1 to 9), anti-RPA ELISA-positive immunoprecipitation-negative sera (lanes 10 to 12), or control sera (normal human serum (NHS), lanes 13 and 14). H, mouse IgG heavy chain.