Figure 2
Characterization of the regions of SRP54 recognized by the human autoantibodies. (a) Summary of the immunoprecipitation of SRP54 and its fragments by the autoantibodies. A restriction map of the plasmid encoding SRP54 is shown at the top (cf [17]): S, SphI; H, HindIII; E, EcoRI; Ba, BamHI; Bg, BglII. The complete SRP54 protein and truncated derivatives are outlined underneath. The names of the polypeptides are shown on the left, and their size in amino acids and the immunoreactivity with the patient autoantibodies is indicated on the right. (b) Immunoprecipitation of SRP54 and its separate domains by sera 19-1 and 25-1 autoantibodies affinity-purified on an amino-terminal SRP54 fragment (amino acids 1–166). SRP54, SRP54N, SRP54G and SRP54M domains were synthesized as 35S-radiolabelled polypeptides in vitro and an aliquot loaded directly onto the gel (Tot). Other aliquots were immunoprecipitated under native conditions with 1 μl serum (Ser), with 1 μl flow-through from the affinity column (Sup) or with 1 μg affinity-purified IgG (Aff). Samples were analysed by SDS-PAGE on 10–15% gels and by fluorography. The estimated molecular masses of the proteins were 54 kDa (SRP54), 23 kDa (SRP54G domain), 21 kDa (SRP54M domain) and 12 kDa (SRP54N domain).