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Bing HEK293 cells (American Type Culture Collection code CRL 11554) were maintained and transfected with pcDNA3 expression vectors by the calcium phosphate method as described (15).
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125I-labeled chemokine and varying concentrations of unlabeled chemokine or gp120, in the presence or absence of 100 or 200 nM sCD4, in a total volume of 100 μ. in binding buffer. Binding reactions were performed in duplicate and incubated at 37°C for 30 min in microcentrifuge tubes. Cells were pelleted and washed in 600 μl of binding buffer containing 0.5 M NaCl. Cell pellets were immersed in scintillation fluid overnight and counted.
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125I-labeled SIVmac239 gp120 for chemokine. Where indicated, 5 μg of mAb was included in the incubation mixture. Reactions were incubated, washed, and counted as in (72).
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We thank R. Sweet for sCD4 and the Drosophila expression system for production of envelope glycoproteins, and R. Desrosiers for the SIVmac239 env gene sequences. Supported by NIH grants AI41581 (J.S. and C.G.), HL51366 (C.G. and N.P.G.), HL36162 (N.P.G.), AI32375 (J.R.), NCI grant CA09382 (L.M.), the Institute Superiore di Sanitá and the University of Padua (LM.), the Rubenstein/Cable Fund at the Perlmutter Laboratory, and gifts from the late William McCarty-Cooper, the G. Harold and Leila Y. Mathers Charitable Foundation, and the Friends 10. The Dana-Farber Cancer Institute is the recipient of a Center for AIDS Research grant (AI-28691) and a Cancer Center grant (CA-06516) from NIH.
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