Figure 1

Anti-U1-70K and anti-70K^apop detection by western blotting. Apoptosis was induced in Jurkat cells by incubation with anisomycin for 8 hours. Western blots were prepared with the resulting cell extracts, and the positions of relevant polypeptides were revealed with patient sera and monoclonal antibodies with the use of a chemiluminescent detection procedure. The positions of the various proteins are indicated on the left, and molecular mass marker positions on the right. (a) U1-70K (70K) detected with a serum from MCTD patient B16 (lanes 1 and 5), anti-70K monoclonal antibody 2.73 (lanes 2 and 6) and an anti-70K single-chain recombinant antibody (scFv; lanes 3 and 7) (70K); lanes 4 and 8, Sm-B/B' detected with a monoclonal anti-Sm-B/B' antibody (ANA125); the position of U1A, which is also recognized by serum from MCTD patient B16 (lanes 1 and 5), was determined by a U1A-specific monoclonal antibody (not shown). In apoptotic cells (lanes 5–8), 70K is present as a 40 kDa species (70K^apop). (b) A serum sample from MCTD patient B16 was applied at 5000-fold (lane 1), 10,000-fold (lane 2) and 20,000-fold (lane 3) dilution on a western blot containing a mixture of non-apoptotic and apoptotic Jurkat cell extracts. In lane 4 the 70K protein was detected with mouse monoclonal antibody 2.73, which reacts much more efficiently with 70K than with 70K^apop.