Figure 2

Characterization of human anti-TIF1β positive sera. A. IP-western blot. Extract from 5 × 10⁶ K562 cells was immunoprecipitated by mouse anti-TIF1β mAb, human anti-TIF1β positive sera (lanes 1 to 3 correspond to case 1 to 3, case 3 is a weakly positive sample), human anti-p155/140 (TIF1-γ/α) positive serum, or normal human serum (NHS). Purified proteins were fractionated by 8% SDS-PAGE, transferred to nitrocellulose filter and probed with mouse anti-TIF1β mAb. Only 1/10 amount of immunoprecipitates was loaded for mAb, case 1, and 2 in order to obtain more comparable signals to case 3. B. Anti-TIF1β antigen-capture ELISA. Four human anti-TIF1β positive sera (left) and controls (right, 3 anti-TIF1-γ, 3 anti-Mi-2, and 2 normal human sera, NHS) were serially diluted from 1:500 and tested by antigen-capture ELISA. C. Western blot. D. Immunofluorescence staining of HEp-2 cells. HEp-2 slides were stained with anti-TIF1β mouse monoclonal antibodies (a), anti-TIF1β antibody positive human autoimmune sera (b-e), or normal human serum (f). Serum dilution 1: 80; anti-TIF1β mAb, 1 μg/ml. ELISA, enzyme-linked immunosorbent assay; NHS, normal human serum; TIF, transcription intermediary factor.