Figure 3
Distinct autoantibodies against the SRP54N and SRP54G domains can both inhibit secretory protein translocation into the ER. (a) The secretory protein preprolactin (PPL) was synthesized as a 35S-radiolabelled precursor in vitro in the presence of salt-washed rough microsomes (RM) and signal recognition particle(SRP) that was preincubated with affinity-purified autoantibodies from sera 19-1 or 25-1 on SRP54 amino acids 1–166 (Aff), with the flow-through fractions (Sup) or with buffer alone. SRP without preincubation (lanes 11 and 12) and translations without added SRP (lanes 13 and 14) or RM (lanes 15 and 16) were used as positive and negative controls, respectively. Samples were treated with proteinase K or not (+ or - PK) and were analysed by SDS-PAGE on 10–15% gels and by fluorography. (b) Serum 25-1 contains two distinct anti-SRP54 activities. SRP54 was synthesized as a 35S-radiolabelled protein in vitro and either digested with V8 protease (+ V8) or incubated in the absence of protease (- V8) as described previously [17]. An aliquot of both digested and undigested material was loaded onto the gel directly (Tot). Both digested and undigested material were immunoprecipitated using 1 μl serum 25-1 (Ser), using 1 μl affinity-column flow-through (Sup) or using 1 μg affinity-purified autoantibodies from serum 25-1 (Aff). Samples were analysed by SDS-PAGE on 10–15% gels and by fluorography.