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. 2006 Apr;12(4):547–554. doi: 10.1261/rna.2302706

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FIGURE 1. — Antibodies in the serum of a primary biliary cirrhosis patient react with P-bodies and stress granules. (Panel I) (A,E) Patient 0080’s serum contained antibodies that reacted with 5–20 dots in the cytoplasm of Hep-2 cells as determined by indirect immunofluorescence. The patient’s serum also contained antibodies directed against E2-pyruvate dehydrogenase complex (E2-PDC), which produced granular, filamentous (mitochondrial) cytoplasmic staining. (C) Treatment of Hep-2 cells with cyclohexamide resulted in disappearance of cytoplasmic dots with no effect on the staining of mitochondria. (E) Patient antibodies colocalized with (F) anti-DCP1a antibodies. To examine the effect of stress on the cellular location of the P-body antigen, cells were treated with arsenite and stained with (I) human serum and (J) anti-TIA antiserum. Human serum 0080 reacted with one or more proteins that colocalized with TIA in stress granules. Overlap of red and green staining is shown in yellow in G and K. DAPI staining in B, D, H, and L indicates the location of cell nuclei. (Panel II) Immunoblotting was used to characterize the putative P-body/stress granule autoantigen. (A) Antibodies in patient 0080’s serum reacted with 70-kDa (E2-PDC) and 60-kDa proteins in an extract prepared from Hep-2 cells. In addition, the serum reacted weakly with a 50-kDa band, which may be a breakdown product of one of the larger proteins (lane 1). Serum from patient 0012 reacted with the 60-kDa protein, but not with E2 PDC. Serum from a third patient (Ge) reacted with a 160-kDa protein, previously shown to be Ge-1, as well as E2 PDC. In addition, Ge serum produced weak bands at 60 kDa and 50 kDa. Serum from patient 0050 reacted with Ge-1 and an unknown 110-kDa protein (a black arrow indicates the location of this protein). (B) To determine the cellular location of the 110-kDa protein, serum 0050 was used to probe an immunoblot prepared using extracts from cytoplasmic (Cyt) or nuclear (Nuc) fractions of Hep-2 cells. Ge-1 was detected in the cytoplasmic fraction, and the unidentified 110-kDa autoantigen was present in the nuclear fraction.

FIGURE 1. — Antibodies in the serum of a primary biliary cirrhosis patient react with P-bodies and stress granules. (Panel I) (A,E) Patient 0080’s serum contained antibodies that reacted with 5–20 dots in the cytoplasm of Hep-2 cells as determined by indirect immunofluorescence. The patient’s serum also contained antibodies directed against E2-pyruvate dehydrogenase complex (E2-PDC), which produced granular, filamentous (mitochondrial) cytoplasmic staining. (C) Treatment of Hep-2 cells with cyclohexamide resulted in disappearance of cytoplasmic dots with no effect on the staining of mitochondria. (E) Patient antibodies colocalized with (F) anti-DCP1a antibodies. To examine the effect of stress on the cellular location of the P-body antigen, cells were treated with arsenite and stained with (I) human serum and (J) anti-TIA antiserum. Human serum 0080 reacted with one or more proteins that colocalized with TIA in stress granules. Overlap of red and green staining is shown in yellow in G and K. DAPI staining in B, D, H, and L indicates the location of cell nuclei. (Panel II) Immunoblotting was used to characterize the putative P-body/stress granule autoantigen. (A) Antibodies in patient 0080’s serum reacted with 70-kDa (E2-PDC) and 60-kDa proteins in an extract prepared from Hep-2 cells. In addition, the serum reacted weakly with a 50-kDa band, which may be a breakdown product of one of the larger proteins (lane 1). Serum from patient 0012 reacted with the 60-kDa protein, but not with E2 PDC. Serum from a third patient (Ge) reacted with a 160-kDa protein, previously shown to be Ge-1, as well as E2 PDC. In addition, Ge serum produced weak bands at 60 kDa and 50 kDa. Serum from patient 0050 reacted with Ge-1 and an unknown 110-kDa protein (a black arrow indicates the location of this protein). (B) To determine the cellular location of the 110-kDa protein, serum 0050 was used to probe an immunoblot prepared using extracts from cytoplasmic (Cyt) or nuclear (Nuc) fractions of Hep-2 cells. Ge-1 was detected in the cytoplasmic fraction, and the unidentified 110-kDa autoantigen was present in the nuclear fraction.