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The bait for the yeast two-hybrid screen was a LexA fusion protein containing the cytoplasmic tail of the mouse CD40 receptor (from residue 219 to the COOH-terminus)
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5 individual clones, with an average insert size of 0.9 kb.
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Abbreviations for the amino acid residues are A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, lle; 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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The C26 cDNA fragment was cut with Eco RI-Hinc III from yeast vector YSD, ligated into Bluescript IISK+ (Stratagene), and then recloned in-frame into the pEBVHisA vector (Invitrogen), with the use of Bam HI and Kpn I cuts, to create pEBVHis/C26. Stable Ramos cell lines containing either this construct or the control construct (pEBVHis/lacZ) were isolated by electroporation and hygromycin selection.
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41
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4 293.CD40L. cells. In some cases, mAb 5C8 (anti-CD40L) was added. Cells were then washed and incubated with saturating concentrations of mAb Leu-16 (anti-CD20) conjugated to fluorescein isothiocyanate (Becton Dickinson) and mAb to CD23 conjugated to phycoerythrin (Biosource International) for 45 min at 4°C in the presence of heat-aggregated IgG (80 μg/ml) (International Enzyme). Cells were washed to remove unbound antibody before fluorescence intensity was measured on a FACSCAN cytofluorograph (Becton Dickinson) with Consort 30 software.
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We thank P. Svec and T. Hawkins for their help in sequencing and J. Jacob for critical reading of the manuscript. G.G. and Z.-Y. are recipients of an Irvington House Institute fellowship and a Leukemia Society special fellowship, respectively. S.L. is an Arthritis Investigator of the Arthritis Foundation. D.B. is an American Cancer Society Research Professor. A.M.C. is supported by the Medical Scientist Training Program grant 5-T32-GM07367. S.L., A.M.C., and D.I.H. are also supported in part by Biogen, Cambridge, MA. This work was supported by National Institute of Allergy and Infectious Diseases grant A122346 (D.B.) and by National Cancer Institute (NCI) grant R01-CA55713 to S.L.
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