Józefa Wȩsierska-Gądek, Rudolf Grimm, Eva Hitchman, Edward Penner

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Members of the glutathione S-transferase gene family are antigens in autoimmune hepatitis Józefa Wȩsierska-Gądek, Rudolf Grimm, Eva Hitchman, Edward Penner Gastroenterology Volume 114, Issue 2, Pages 329-335 (February 1998) DOI: 10.1016/S0016-5085(98)70485-8 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Analysis of immune complexes formed by four anti-SLA–positive sera during immunodiffusion. Proteins separated on 10% SDS gel were (A) stained with Coomassie blue R-250 or blotted onto membrane, and (B) the biotinylated antigens were detected after incubation with streptavidin-HRP by chemiluminescence. Molecular weight markers are as follows: myosin, 200 kilodaltons; β-galactosidase, 116 kilodaltons; phosphorylase b, 97 kilodaltons; bovine serum albumin, 66 kilodaltons; glutamic dehydrogenase, 55 kilodaltons; lactate dehydrogenase, 36 kilodaltons; carbonic anhydrase, 31 kilodaltons; and trypsin inhibitor, 21 kilodaltons. Gastroenterology 1998 114, 329-335DOI: (10.1016/S0016-5085(98)70485-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Analysis of immune complexes formed by four anti-SLA–positive sera during immunodiffusion. Proteins separated on 10% SDS gel were (A) stained with Coomassie blue R-250 or blotted onto membrane, and (B) the biotinylated antigens were detected after incubation with streptavidin-HRP by chemiluminescence. Molecular weight markers are as follows: myosin, 200 kilodaltons; β-galactosidase, 116 kilodaltons; phosphorylase b, 97 kilodaltons; bovine serum albumin, 66 kilodaltons; glutamic dehydrogenase, 55 kilodaltons; lactate dehydrogenase, 36 kilodaltons; carbonic anhydrase, 31 kilodaltons; and trypsin inhibitor, 21 kilodaltons. Gastroenterology 1998 114, 329-335DOI: (10.1016/S0016-5085(98)70485-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Immunoprecipitation of rat liver cytosol proteins by anti-SLA–positive sera. Immune complexes purified on Protein G–Sepharose and separated on 15% SDS gel were transferred onto nitrocellulose. (A) Proteins were stained by Ponceau S, and (B) biotinylated antigens were detected autoradiographically after overlay with streptavidin-HRP by chemiluminescence. Molecular weight markers are as follows: bovine serum albumin, 66 kilodaltons; glutamic dehydrogenase, 55 kilodaltons; lactate dehydrogenase, 36 kilodaltons; carbonic anhydrase, 31 kilodaltons; trypsin inhibitor, 21 kilodaltons; lysozyme, 14 kilodaltons; and aprotinin, 6 kilodaltons. For control, human serum was omitted during incubation or normal serum of healthy volunteers was used. Gastroenterology 1998 114, 329-335DOI: (10.1016/S0016-5085(98)70485-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Testing of anti-SLA–positive sera for reactivity with antigens of rat liver cytosol. Normal sera obtained from healthy individuals (lanes 10–13). Blot strips were incubated with patient sera (1:500) and with anti-human Ig coupled to HRP (1:2000).16 Lanes 1 and 2, Coomassie blue R-250 (CB) staining. Lane 1, molecular weight markers: see Figure 1. Lanes 2–13, SLA. Gastroenterology 1998 114, 329-335DOI: (10.1016/S0016-5085(98)70485-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Purification and analysis of rat liver cytosol and GSTs. (A) Coomassie blue R-250 stained 10% SDS gel revealing the fractionation of rat liver homogenate. (B) N-terminal sequence (single letter code for amino acids) of the reactive protein bands of rat liver cytosol (top to bottom). Gastroenterology 1998 114, 329-335DOI: (10.1016/S0016-5085(98)70485-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Purification and analysis of rat liver cytosol and GSTs. (A) Coomassie blue R-250 stained 10% SDS gel revealing the fractionation of rat liver homogenate. (B) N-terminal sequence (single letter code for amino acids) of the reactive protein bands of rat liver cytosol (top to bottom). Gastroenterology 1998 114, 329-335DOI: (10.1016/S0016-5085(98)70485-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Immunoblotting using affinity-purified GSTs as antigen. Isolated GSTs were resolved on 15% SDS gel. CB, Coomassie blue R-250. Gastroenterology 1998 114, 329-335DOI: (10.1016/S0016-5085(98)70485-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Reactivity of anti-SLA–positive serum with affinity-purified GSTs after two-dimensional PAGE. (A) Ponceau S staining; (B) immunoblotting. IEF, isoelectric focusing. Gastroenterology 1998 114, 329-335DOI: (10.1016/S0016-5085(98)70485-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Reactivity of anti-SLA–positive serum with affinity-purified GSTs after two-dimensional PAGE. (A) Ponceau S staining; (B) immunoblotting. IEF, isoelectric focusing. Gastroenterology 1998 114, 329-335DOI: (10.1016/S0016-5085(98)70485-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions