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Volumn 279, Issue 5358, 1998, Pages 1961-1964

Dependence of BSAP repressor and activator functions on BSAP concentration

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EID: 0032549828     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5358.1961     Document Type: Article
Times cited : (52)

References (31)
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    • note
    • 32P]dCTP (deoxycytidine 5′-triphosphate) and Klenow enzyme (22). The binding reactions with crude nuclear extracts were performed as described (9), with 8 μg of nuclear extract and 4 μg of poly(dI-dC) nonspecific competitor. The protein complexes formed were resolved from free probe by electrophoresis through 5% polyacrylamide gels (29:1) containing 0.25x tris-borate EDTA buffer. Top strands of the oligonucleotides used for competition reactions are as follows: CD19, 5′-CTAGACAGACACCCATGGTTGA-GTGCCCTCCAGT-3′; Iε, 5′-GTTAGCTGAGGGCA-CTGAGGCAGAGCGGCCCCTAGG-3′; JC, 5′-GG-TGTGCGTCTTTCCAGTGTAGCATGCAGTTCAA-3′; and 3′ α E, 5′-CTAGATCACTTCCCTGGGGTG-TTGAGCCACCCAT-3′.
  • 16
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    • note
    • 7 cells in 270 μl of RPMI were transfected at 250 V and 960 μF with a Gene Pulser (Bio-Rad). Transfected cells (1:10 dilution) were all-quoted into 96-well plates and 24 hours later were mixed with an equal volume of 2x G418-sulfate selection medium. G418-resistant clones were visible by microscopy after 2 to 3 weeks of culture. At that time, cells from positive wells were replated at a concentration of less than one cell per well, and positive samples were expanded and maintained in the selection media. For protein immunoblot analyses, nuclear extracts (25 μg) from stably transfected clones were boiled for 5 min, size-fractionated by SDS-polyacrylamide gel electrophoresis (12.5%), and transferred to a nitrocellulose filter. After pretreatment with 5% dry milk in 1x phosphate-buffered saline, the filters were incubated for 3 hours with a 1:10,000 dilution of antibody specific for the BSAP paired domain (gift of M. Busslinger) or antibody specific for γ or α immunoglobulin heavy chain, or κ or λ light chain (Zymed).
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    • 32P]dATP (deoxyadenosine 5′-triphosphate) (22).
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    • note
    • 7 cells in log-phase were transfected with 9 μg of supercoiled test plasmid. Cell extracts were prepared 44 to 48 hours after transfection and assayed for CAT activity (26).
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    • note
    • We thank A. Winoto and W. Sha for helpful comments and critical reading of the manuscript. Supported by the W. M. Keck Foundation and Public Health Service training grant CA09179.


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