NF-AT-driven interleukin-4 transcription potentiated by NIP45

Science

Volumn 274, Issue 5294, 1996, Pages 1903-1905

  Hodge, Martin R ^a   Chun, Hyung J ^b   Rengarajan, Jyothi ^b   Alt, Aya ^b   Lieberson, Rebecca ^b   Glimcher, Laurie H ^b  

^a MILLENNIUM PHARMACEUTICALS INC   (United States)

^b NONE

Author keywords

[No Author keywords available]

Indexed keywords

INTERLEUKIN 4; NUCLEAR FACTOR; PROTEIN; PROTEIN NIP 45; UNCLASSIFIED DRUG;

ANIMAL TISSUE; ARTICLE; CELL STRAIN HEPG2; CONTROLLED STUDY; GENE ACTIVATION; HUMAN; HUMAN CELL; LYMPHOID TISSUE; LYMPHOMA CELL; MOUSE; NONHUMAN; PRIORITY JOURNAL; PROMOTER REGION; PROTO ONCOGENE; TESTIS;

AMINO ACID SEQUENCE; ANIMALS; BASE SEQUENCE; CARRIER PROTEINS; CELL LINE; CELL NUCLEUS; CLONING, MOLECULAR; DNA-BINDING PROTEINS; GENES, REPORTER; HUMANS; INTERLEUKIN-4; INTRACELLULAR SIGNALING PEPTIDES AND PROTEINS; MALE; MOLECULAR SEQUENCE DATA; NFATC TRANSCRIPTION FACTORS; NUCLEAR PROTEINS; PROMOTER REGIONS (GENETICS); RECOMBINANT FUSION PROTEINS; SPLEEN; TESTIS; THYMUS GLAND; TRANS-ACTIVATION (GENETICS); TRANSCRIPTION FACTORS; TRANSFECTION; TUMOR CELLS, CULTURED;

ANIMALIA;

EID: 0030474682     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5294.1903     Document Type: Article

References (25)

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10. A yeast two-hybrid bait was prepared by cloning a 900-bp fragment of murine NF-ATp (9) spanning amino adds 228 to 520 into the Bam HI site of vector pEG202 (22). In-frame fusion of the polypeptide sequence was confirmed by DNA sequence analysis. This bait was used to screen a D10 T cell cDNA library, constructed in the plasmid pJG4-5, for clones encoding interacting polypeptides as described (22).
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13. NIP45Δ was produced by creating a two-base deletion at nucleotide 50. This alteration results in the introduction of missense mutations at amino acid 13 and termination of the polypeptide after an additional 22 residues.
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18. BHK cells were transfected with 1 μg of the indicated plasmids as described (23). Cells were incubated overnight in culture media and either fixed directly or first stimulated with 1 mM ionomycin for 10 min before fixation. Fixed cells were permeabilized (23) and probed with monoclonal antibody 12CA5 to HA (anti-HA) (Boehringer Mannheim) plus indocarbocyanine-labeled donkey anti-mouse (Jackson ImmunoResearch) and then counterstained with the dye Hoechst 33258.
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21. 6 cells in 0.4 ml of phosphate-buffered saline with 5 μg of each plasmid for 10 min at room temperature prior to to electroporation at 975 μF, 280 V. The expression vectors for c-maf (pMEX-Maf/ pREP4), NF-AT (pREP4-NF-ATp), and NIP45 are those used in Fig 4. The control for NIP 45 is the empty vector, pCl. ELISA was done according to the instructions of Pharmingen except with modification as described (3).
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25. We thank E. R. Price and T. McKeon for assistance with immunofluorescence, G. Crabtree and L. Timmerman for generous provision of NF-AT antibodies, Dr. Hong Zhou for providing MHC class II data, and M. Kaplan and I-C. Ho for careful review of the manu-script. Supported by NIH grant AI37833.