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β repertoires were addressed by reverse transcriptase-PCR in PBMCs and exhibited an oligoclonal pattern. Thus, patient P3 was considered leaky.
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note
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RAG-1 and RAG-2 expression vectors used for all recombination assays were identical except for the single mutation introduced; thus, promotor and 5′ and 3′ untranslated region influences on expression are excluded. Cells transfected with RAG expression vectors were boiled in SDS lysis buffer Equal amounts of total protein (100 μg) were fractionated by 10% SDS-polyacrylamide gel electrophoresis. Protein was transferred to nitrocellulose and detected by immunoblotting with affinity-purified antibodies to RAG as described (20). 23. K.S., U.P., D.L., and C.R.B. are recipients of grants of the Sonderforschungsbereich 322 from the Deutsche Forschungsgemeinschaft. G.H.G. is supported by a PHS grant awarded to the Stanford University Program in Career Biology. M.R.L. is a Leukemia Society of America Scholar, and research in his laboratory is supported by grants from the NIH and a grant from the Council for Tobacco Research. S.D. is supported by a grant from the National Cancer Institute and by the Howard Hughes Medical Institute.
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