From: Autoantibody Reactive with RNA Polymerase III in Systemic Sclerosis Ann Intern Med. 1993;119(10):1005-1013. doi:10.7326/0003-4819-119-10-199311150-00007 Figure Legend: Precipitated proteins of [35S]methionine-labeled HeLa cell extract obtained using serum specimens and fractionated on 7.leftright5% ( ) and 12% ( ) polyacrylamide-SDS gel. Lane Mr shows molecular weight markers; lane 1 shows nonspecific proteins precipitated by normal human serum; lanes 2, 3, 4, 5, 6, and 7 show specific proteins precipitated in serum specimens from six patients with systemic sclerosis. The positions of molecular weight standards (in kilodaltons) are shown to the left of lane Mr. Two panels are included to show the separation of proteins with molecular weights of more than 30 kd and less than 40 kd, respectively. Date of download: 9/26/2017 Copyright © American College of Physicians. All rights reserved.

From: Autoantibody Reactive with RNA Polymerase III in Systemic Sclerosis Ann Intern Med. 1993;119(10):1005-1013. doi:10.7326/0003-4819-119-10-199311150-00007 Figure Legend: Serum antibodies from patients with SSc shown by immunoblotting to be reactive with HeLa cell RNA polymerase III.9Table 1[21]Each lane contained partially purified RNA polymerase III obtained from 5 10 HeLa cells by biochemical procedures. The substrate was fractionated on a 11% polyacrylamide-SDS gel and electrophoretically transferred to nitrocellulose sheets. The sheets were incubated with normal serum (lane 1) and four representative serum specimens from groups 1, 2, and 3 (lanes 2-5); the groups are defined in . The IgG bound to proteins was visualized using enzyme-linked immunoassay. Dots beside lanes indicate the positive protein bands. The nitrocellulose strip was also stained with amido-black (lane 6). An arrow to the right of lane 6 indicates the position of each subunit protein, according to the conformity of its molecular weight, as previously described . Four arrowheads to the right of lane 6 indicate the positions of four additional proteins, which are likely contaminants. The positions of molecular weight standards (in kilodaltons) are given to the right of the panel. Date of download: 9/26/2017 Copyright © American College of Physicians. All rights reserved.

From: Autoantibody Reactive with RNA Polymerase III in Systemic Sclerosis Ann Intern Med. 1993;119(10):1005-1013. doi:10.7326/0003-4819-119-10-199311150-00007 Figure Legend: Depletion of RNA polymerase III activity from HeLa S100 extracts by immunoprecipitation.Table 2323Antibodies were coupled to protein-A sepharose and used in depletion experiments. The HeLa S100 extract was preincubated with IgG-coupled protein-A sepharose, obtained using normal human serum (lane 1), four control sera with various antinuclear antibodies (the specificities are marked at the top of lanes), and six representative serum specimens from the three groups (lanes 4-9); see for group classification. We also carried out mock immunoprecipitation (no antibodies coupled to the protein-A sepharose). After removal of the sepharose by brief centrifugation, the supernatant was subjected to the RNA polymerase III transcription reactions using adenovirus pVA I as the DNA template. The [ P]orthophosphate-labeled adenovirus VA I RNA products by the in vitro transcription reactions were determined by 8% polyacrylamide-urea (7 mol/L) gels and visualized by autoradiography. Anti-topo I = anti-topoisomerase I antibody I; anti-U3 RNP = anti-U ribonucleoprotein antibody (fibrillarin). Date of download: 9/26/2017 Copyright © American College of Physicians. All rights reserved.

From: Autoantibody Reactive with RNA Polymerase III in Systemic Sclerosis Ann Intern Med. 1993;119(10):1005-1013. doi:10.7326/0003-4819-119-10-199311150-00007 Figure Legend: Indirect immunofluorescence test for antinuclear antibody using HEp-2 cells as substrate.Top.[16]Bottom.Serum specimens from patients with systemic sclerosis were diluted 1:40 with phosphate-buffered saline and used as the first antibody. Immunofluorescence shows nucleolar staining, suggesting that serum contains antinuclear antibody that is apparently identical to the anti-RNA polymerase I antibody shown with antinucleolar staining by Reimer and colleagues . Immunofluorescence shows nuclear speckled staining without nucleolar staining. Date of download: 9/26/2017 Copyright © American College of Physicians. All rights reserved.