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Volumn 276, Issue 5312, 1997, Pages 604-607

Identification of α-fodrin as a candidate autoantigen in primary Sjogren's syndrome

Author keywords

[No Author keywords available]

Indexed keywords

CYTOSKELETON PROTEIN; FODRIN; GAMMA INTERFERON; INTERLEUKIN 2; SPECTRIN ANTIBODY; UNCLASSIFIED DRUG;

EID: 0030999866     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.276.5312.604     Document Type: Article
Times cited : (398)

References (37)
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    • note
    • The salivary gland homogenates from 3d-Tx NFS/sld mice were prepared in 20 mM tris-HCl buffer (pH 7.2) containing 0.15 M NaCl and proteinase inhibitors (5 mM benzamidine-HCl, 2 mM diisopropyl fluoride, and 2 mM EDTA). The supernatant of the homogenates was fractionated on a Superose 12HR column (Pharmacia) and further purified on a DEAE-Cosmogel column (Nakarai, Japan). SDS-polyacrylamide gel electrophoresis was carried out in 10% gels as described by (28).
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    • note
    • Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
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    • note
    • 3H]thymidine was added per well, and the incorporated radioactivity was determined.
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    • note
    • The amounts of IFN-γ and IL-2 produced were measured by enzyme-linked immunosorbent assay (ELISA) (29) with IFN-γ - specific monoclonal antibodies (Pharmingen) and mlL-2 ELISA system (Amersham).
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    • note
    • A synthetic peptide of α-fodrin corresponding to the identified 20-amino acid residues [RQKLEDSYRFQFFQRDAEEL) (6)] was produced with an Applied Biosystems model 430A peptide synthesizer. Polyclonal rabbit antibody to the synthetic peptide was developed and affinity purified. Rabbit serum to the synthetic peptide was diluted 1:25 when used for protein immunoblots.
  • 14
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    • note
    • For immunohistochemistry, formalin-fixed paraffin-embedded salivary gland sections were deparaffinized in xylen and hydrated in ethanol. Sections were stained with polyclonal rabbit antibody to synthetic α-fodrin peptide diluted 1:500 dilution in 10% (v/v) fetal bovine serum, followed by incubation with biotin-labeled goat serum to rabbit as the linking antibody. After the preparations were rinsed, binding was detected with an avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA). Specificity of the staining was confirmed by the observation that incubating polyclonal rabbit antiserum with synthetic peptide completely blocked staining.
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    • note
    • JS-1 cDNA encoding the human nonerythroid α-fodrin cDNA was constructed by inserting cDNA (base pairs 1 through 1784) into the Eco RI site of plasmid pGEX-2T (Pharmacia). GST fusion protein was expressed and purified with a GST gene fusion system (Pharmacia) as described (30). Protein immunoblots were performed and proliferative T cell responses were determined as described in the legend to Fig. 1.
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    • note
    • 3H]thymidine was added per well. Incorporation was measured by liquid scintillation counting.
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    • note
    • We thank S. Sone and K. Hoil for human sera and R. T. Moon for cDNA. Supported by grants-in-aid for scientic research from the Ministry of Education, Science, and Culture of Japan.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.