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C.-Y. Chen, unpublished results.
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Chen, C.-Y.1
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2642613389
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note
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Various regions of IL-2 cDNA were deleted. Del 1 was constructed by creating an Eco RV site at codon 4 and codon 5 of IL-2 with PCR-based method to obtain pβACT-IL2RV. The Hind III to Eco RV fragment was replaced with an oligonucleotide that restored the start codon of IL-2. Del 2 was constructed by removing a Eco RV to Afl II fragment from pβACT-IL2RV and replacing it with a PCR fragment digested with Apa LI/Afl II, in which nt 67 to 114 were removed. Del 3 was constructed by subcloning two PCR fragments - nt 1 to 130, which had been digested with Hind III and Bgl II, and nt 200 to 350, which had been digested with Bgl II and Afl II - between the Hind III and Afl II sites of pβACT-IL2. Del 4 and Del 5 were constructed by digesting pβACT-IL2 with Afl II and Stu I, and with Stu I and Sty I, respectively, and then religating with T4 DNA ligase. Del 6 was constructed by removing a Sty I-Kpn I fragment pβACT-IL2 and replacing it with an oligonucleotide containing the polyadenylation signal of IL-2. None of the IL-2 mutants altered the reading frame.
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44
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2642646799
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note
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The entire 3′ UTR, including the polyadenylation signal of human c-fos or GM-CSF amplified by RT-PCR, and a chemically synthesized double-stranded oligonucleotide containing two nonamers, UUAUUUAUU-gauccUUAUUUAUU, and a polyadenylation signal were subcloned between the Stu I and Kpn I sites of pβACT-IL2. CAT reporters were constructed by sub-cloning the entire coding region of the CAT gene, amplified by PCR, between the Hind III and Stu I sites of pβACT-IL2 to obtain CAT-3′ UTR (IL2) or between the Bgl II and Stu I sites of Del 3 to obtain 5′ UTR (IL2)-CAT-3′ UTR (IL2), in which the reading frame of CAT is in-frame with that of IL-2.
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2642639320
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note
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63-c-Jun (KM-1, Santa Cruz Biotechnology), and the antibody-antigen complexes were visualized by enhanced chemiluminescence (Amersham). The membrane was stripped and reprobed with an antibody to c-Jun (N-G, Santa Cruz Biotechnology).
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note
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We thank M. S. Barbosa for constitutively active MKK6, T. Kallunki for JNK2(GE), A. S. Kiselyov for synthesizing SB202190, and C. Moroni for helpful discussions. C.-Y.C., F.K., and Z.W. were supported by postdoctoral fellowships from the Leukemia Research Foundation and the Tobacco-Related Disease Research Program, Association pour la Recherche sur le Cancer, and Human Frontiers Science Project, respectively.
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