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L domains of the NIP-specific antibody B1-8, followed by a CD8α linker region and the CD3ζ transmembrane part, and carries a Bam HI cloning site after the CD3ζ sequence. Bam HI fragments of the wild-type or mutated cytoplasmic γ2am and mb-1 coding sequences were obtained by PCR and inserted into psc-cyt to yield the expression vectors pANPCD8γ2am, pANPCD8γ2amY20L, pANPCD8γ2amY20L,M23L, pANPCD8mb-1, and pANPCD8mb-1M1. The pANPCD8tl vector encodes a scAgR with a short cytoplasmic sequence of the five amino acids RRIDP. The vectors were linearized with Pvu I and introduced into K46 and K46λ 12 cells by electroporation.
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Müller, R.1
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1842322595
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note
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Single-letter abbreviations for the amino acid residues are as follows: D, Asp; G, Gly; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; R, Arg; S, Ser; V, Val; X, any amino acid; and Y, Tyr.
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1842350422
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note
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Transfected cells were surface-biotinylated and lysed with 1% digitonin buffer as described (5). After a preclearing step with Sepharose beads, NIP-specific molecules were immunoprecipitated from the lysates with the use of NIP-Sepharose or Sepharose as control. After being washed three times with the 1% digitonin buffer, the NIP-Sepharose beads were boiled in reducing SDS sample buffer. The released proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and electroblotted onto a nitrocellulose membrane. Biotinylated proteins were detected with Str-HRP and the ECL detection system (Amersham, Braunschweig, Germany).
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12
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1842283354
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note
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450. Mean values and standard deviations from triplicates were then used to plot the dose-response curves. The statistical relevance of each mean value was verificated by use of Student's t test. Apart from transfectant K46λγ2amY20L, two or three independent clones of each transfectant were analyzed. B cells were propagated in RPMI 1640 medium containing 10% fetal calf serum (FCS) ( Vitromex, Vilshofen, Germany) and G418 (0.5 mg/ml); the T cells were cultured in DMEM containing 10% FCS and, in case of the CTLL cells, 5% of supernatant of the IL-2-producing transfectant X63-IL-2.
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We thank L. Nitschke, M. C. Lamers and K. Rajewsky for communication of unpublished data; T. Brocker, K. Karjalainen, and T. Simon for single-chain vectors; and L. Leclercq and P. Nielsen for critical reading of this manuscript. Supported partly by grants from the Deutsche Forschungsgemeinschaft (Re 571-3-1 and SFB 364).
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