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HEK 293 cells were transected with plasmids encoding DRα, DRβ, Ii, DMA, and DMB individually. Transfectants were selected in media containing G418, hygromycin, and puromycin. Expression of human leukocyte AG DR (HLA-DR) was evaluated by fluorescence-activated cell sorting analysis with either monoclonal antibodies to HLA-DR1 or HLA-DR4 (One Lambda, Canoga Park, CA). Expression of DMA, DMB, and Ii was detected by protein immunoblot with antibodies to DMA (anti-DMA), anti-DMB, and anti-li.
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0344056138
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note
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The constructs pHG810, plHG812, plHG813, and plHG814 were made as follows. The Ii fragments (amino acids 1 to 60, 1 to 80, and 1 to 108) were generated by polymerase chain reaction amplification with specific sets of primers and cloned into the eukaryotic expression vector pcDNA3.1 (Invitrogen, San Diego, CA) to produce the pl60, pl80, and pl108 plasmids, respectively. A 400-base pair DNA fragment containing the HA(307-319) epitope was amplified from a plasmid encoding an influenza virus HA and was inserted into pl60, pl80, and pl108 to generate plH60, plH80, and plH108, respectively. A DNA fragment encoding enhanced green fluorescence protein (EGFP) from pEGFP-N3 (Clontech, Palo Alto, CA) was then fused in-frame to the COOH-terminus of the 1H fusion constructs. The resultant plasmids were named plHG812, plHG813, and plHG814, pHG810 was constructed in the same way except for omission of the Ii fragment.
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0344918479
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Supplementary material is available at
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Supplementary material is available at www.science. org/feature/data/987217.shl
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note
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Total RNAs were extracted from 1558mel and 1359mel with Trizol reagent from GIBCO BRL (Gaithersburg, MD). Polyadenylated RNAs were purified from total RNA by using polyATract System (Pro-mega, Madison, WI) and converted to cDNA with a cDNA construction kit (GIBCO BRL) with an oligo-dT primer. The cDNA inserts were cloned into the expression vector pTSX. The cDNA libraries were electroporated into DH10B bacteria cells (GISCO BRL). Plasmid DNA pools were prepared from bacteria, each consisting of about 100 cDNA clones.
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31
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0344056136
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note
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5 cDNA clones were screened by expression in 293IMDR4 cells (17). Two positive pools were identified in the first screening. Individual DNA was prepared from about 800 Escherichia coli colonies transformed with the positive-pool DNA, and six positive cDNA clones were isolated by repeating the screening procedure.
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DNA transfection with GM-CSF assays were performed as previously described [R. F. Wang, P. F. Robbins, Y. Kawakami, X. Q. Kang, S. A. Rosenberg, J. Exp. Med. 181, 799 (1995); R.-F. Wang et al., ibid. 184, 2207 (1996); R.-F. Wang et al., J. Immunol. 161, 3596 (1998)].
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Peptides were synthesized by a solid-phase method with an automatic peptide synthesizer (Model AMS 422, Gilson, Worthington, OH). The masses of some peptides were confirmed by mass spectrometry, and peptides were purified by high-pressure liquid chromatography as needed to obtain purities greater than 98%.
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44
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0345349739
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note
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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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Cell lysates were prepared by lysis buffer. Equal amounts of total protein were loaded for each sample and separated on a 4 to 16% SDS-polyacrylamide gel by electrophoresis (SDS-PAGE), The proteins were then blotted onto a nitrocellulose membrane and incubated with a murine antibody to human CDC27 (Transduction Lab) or a rabbit polydonal antibody to human CDC27. After incubation with an anti-mouse or anti-rabbit immunoglobulin G conjugated with horseradish peroxidase, protein was detected with the ECL system (Amersham).
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0345349738
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note
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We thank J. Yannelli, J. R. Wunderlich, and the TIL lab for cell lines and reagents; M. Parkhurst and E. Fitzgerald for peptide synthesis; R. N. Germain for discussion and reading of the manuscript; E. Long for the plasmid containing human invariant chain cDNA; P. Cresswell for antibodies to DM and to Ii; C.-H. Chang for cDNA encoding DMA and DMB; J. Trowsdale for an antibody to DM; and P. Hieter for antibodies to CDC27 AND CDC16.
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