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c are the total number of particles counted and the total number of cells used, respectively.
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50
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32P]SPP was quantitated by scintillation counting. Specific binding was calculated as total binding minus binding in the presence of 100-fold excess of unlabeled SPP.
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51
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The enhanced GFP cDNA (Clontech, Palo Alto, CA) was fused at the COOH-terminus of the EDG-1 cDNA, and the chimera was cloned into a eukaryotic expression vector containing a cytomegalovirus promoter (pEGFP). Expression of the correct polypeptide was confirmed by DNA sequencing as well as by immunoblot analysis of transfected cells. HEK293 clones stably expressing EDG-1-GFP were isolated by standard procedures (12).
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We thank R. D. Berlin and T. Maciag for support and discussions and T. I. Bonner for sharing unpublished results. Supported by research grants from the NIH to T.H. (DK45659 and HL49094) and S.S. (GM43880 and CA61774) and by a grant from the American Cancer Society (BE-275) to S.S.
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