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Journal of Lipid Research, Vol. 43, 2042-2048, December 2002
Copyright © 2002 by Lipid Research, Inc.

Structural determinants of sphingolipid recognition by commercially available anti-ceramide antibodies

L. Ashley Cowart, Zdzislaw Szulc, Alicja Bielawska and Yusuf A. Hannun¹

Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425

¹ To whom correspondence should be addressed. e-mail: hannun{at}musc.edu

The sphingolipid mediator ceramide is involved in cellular processes such as apoptosis, differentiation, responses to cytokines, and stress responses. Experimental evidence suggests that the intracellular location of ceramide may be a key factor in determining its ultimate cellular effects. One approach to ceramide localization is the use of recently developed anti-ceramide antibodies for immunocytochemical studies. Two such commercial preparations are now available; we sought to compare and contrast their specificity for ceramide and/or other cellular lipids. By using lipid overlay assays and a diverse panel of sphingolipids, we were able to delineate the specificity and thus, the utility of these reagents. Our results indicate that one of these anti-ceramide preparations is quite specific for ceramide and dihydroceramide, whereas the other preparation recognizes dihydroceramide, phosphatidylcholine, and sphingomyelin.

Furthermore, through the use of chemically modified ceramides in similar assays, we were able to determine some structural determinants of lipid recognition by both of these reagents.

Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; SMase, sphingomyelinase

Supplementary key words ceramide • antibody • lipid-antibody interaction • sphingolipid • lipid • lipid chemistry • signal transduction

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