Volume 117, Issue 4, Pages 823-830 (October 1999) Identification of an autoimmune enteropathy–related 75-kilodalton antigen  Ichiro Kobayashi, Keisaku Imamura, Mitsuru Kubota, Susumu Ishikawa, Masafumi Yamada, Hidefumi Tonoki, Motohiko Okano, Wulf B. Storch, Tetsuya Moriuchi, Yukio Sakiyama, Kunihiko Kobayashi  Gastroenterology  Volume 117, Issue 4, Pages 823-830 (October 1999) DOI: 10.1016/S0016-5085(99)70340-9 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) cDNA and deduced amino acid sequences of AIE-75. Thick lines indicate PDZ domains. Dashed line and thin solid line indicate short segments similar to SH-3 and GK domains, respectively. Nucleotide sequence predicted from the genomic DNA sequence is shown in lowercase letters. (B) Exon-intron structure of AIE-75 gene. Vertical lines indicate exons. Gastroenterology 1999 117, 823-830DOI: (10.1016/S0016-5085(99)70340-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) cDNA and deduced amino acid sequences of AIE-75. Thick lines indicate PDZ domains. Dashed line and thin solid line indicate short segments similar to SH-3 and GK domains, respectively. Nucleotide sequence predicted from the genomic DNA sequence is shown in lowercase letters. (B) Exon-intron structure of AIE-75 gene. Vertical lines indicate exons. Gastroenterology 1999 117, 823-830DOI: (10.1016/S0016-5085(99)70340-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Immunoblot analysis of AIE-75 in tissue homogenates. Homogenates from the tissues listed were electrophoresed on 8% SDS-polyacrylamide gel under reducing conditions and were immunoblotted with rabbit anti–MBP-D6 serum or control anti-MBP serum. Arrow indicates a 75-kilodalton immunoreactive band. Gastroenterology 1999 117, 823-830DOI: (10.1016/S0016-5085(99)70340-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Inhibition study with the fusion protein MBP-K2. Jejunal (lanes 1 and 2) and renal (lanes 3 and 4) tissue homogenates were electrophoresed on 7.5% SDS-polyacrylamide gel under reducing conditions and were immunoblotted with AIE serum that had been pretreated with 500 μg/mL of MBP-K2 (lanes 2 and 4) or MBP (lanes 1 and 3). Note that the immunoreactive 75-kilodalton band (arrow) was abolished by pretreatment of the serum with MBP-K2. Gastroenterology 1999 117, 823-830DOI: (10.1016/S0016-5085(99)70340-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 In vitro transcription and translation. Rabbit reticulocyte lysate incubated with pcDNA 3 mock vector (lane 1) or pcDNA-AIE-75 (lane 2) was immunoblotted with the serum from a patient with AIE. The mobility of in vitro–translated AIE-75 was comparable with that of the 75-kilodalton antigen (arrow) in the jejunal lysate (lane 3). Several bands other than that at 75 kilodaltons represent the reactions of the tissue components with HRP–anti-human IgG (lane 4). Gastroenterology 1999 117, 823-830DOI: (10.1016/S0016-5085(99)70340-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 Immunoblotting of in vitro–translated AIE-75 with AIE and control sera showing reactivity of in vitro–translated AIE-75 with the sera from (A) patients with AIE (AIE 1-4) or (B) patients with enteritis of unknown cause, ulcerative colitis (UC), Crohn's disease (CD), and Behçet's disease involving the small intestine. Gastroenterology 1999 117, 823-830DOI: (10.1016/S0016-5085(99)70340-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 Immunoblotting of in vitro–translated AIE-75 with AIE and control sera showing reactivity of in vitro–translated AIE-75 with the sera from (A) patients with AIE (AIE 1-4) or (B) patients with enteritis of unknown cause, ulcerative colitis (UC), Crohn's disease (CD), and Behçet's disease involving the small intestine. Gastroenterology 1999 117, 823-830DOI: (10.1016/S0016-5085(99)70340-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 Northern blot analysis. Hybridization of RNA extracted from various human organs was performed with use of [α-32P]deoxycytidine triphosphate–labeled D6 cDNA as a probe. PBMC, peripheral blood mononuclear cells. Arrow indicates a single transcript of 2.3 kb. Hybridization with β-actin probe is shown at bottom. Gastroenterology 1999 117, 823-830DOI: (10.1016/S0016-5085(99)70340-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 7 Immunohistochemical localization of AIE-75. Affinity-purified anti–MBP-D6 was used for staining sections of the (A and B) ileum, (C) colon, and (D) kidney (original magnifications: A, 40×; B, 200×; C, 20×; D, 40×). Gastroenterology 1999 117, 823-830DOI: (10.1016/S0016-5085(99)70340-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions