FIGURE 1.

Antibodies in the serum of a primary biliary cirrhosis patient react with a novel P-body component. (Panel I) Patient 0020 serum contained antibodies that reacted with 5–20 dots in the cytoplasm of Hep-2 cells as determined by indirect immunofluorescence (I-A,E). The patient’s serum also contained antibodies directed against E2-pyruvate dehydrogenase complex (E2-PDC), which produced a cytoplasmic, linear staining pattern, and antibodies directed against an unknown antigen that was present diffusely throughout the nucleus. Treatment of Hep-2 cells with cyclohexamide resulted in disappearance of cytoplasmic dots with little effect on the staining of mitochondria or nuclei (I-C). (I-E) Patient antibodies (green) co-localized with (I-F) anti-DCP2 antibodies (red). To further confirm that patient antibodies reacted with P-bodies, Hep-2 cells were transfected with a plasmid encoding the Flag-epitope fused to P-body component DCP1a. Cells were fixed and stained with (I-I) patient serum (green) and (I-J) anti-Flag antibodies (red). (I-G,K) Overlap of red and green staining is shown in yellow. DAPI staining in I-B,D,H,L indicates the location of cell nuclei. (Panel II) Immunoblotting was used to characterize the putative P-body autoantigen. Antibodies in patient serum reacted with 70-kDa (E2-PDC) and ~160-kDa proteins in an extract prepared from Hep-2 cells. (II-A) Normal human serum did not react with either of these two proteins. To specifically test the patient serum for anti-GW182 antibodies, recombinant GST-GW182 was prepared, fractionated by PAGE, and transferred to nitrocellulose membranes. (II-B) Rabbit anti-GW182 antiserum reacted strongly with the recombinant protein and with associated breakdown products. Neither patient serum nor normal human serum contained antibodies directed against GW182. Both human sera reacted with an ~80-kDa protein in the extract that is likely to be residual, contaminating E. coli protein.