Sperm - Egg Binding Protein or Proto - Oncogene ?

tized. The hybridization signal of the radiolabeled probe appears as white grains. All specimens were observed under dark field illumination after nuclear counterstain with hematoxylin. Immunostaining for factor Vill-related antigen (4) confirmed that injury was limited to endothelium. 23. Binding was carred out in buffer containing 50 mM NaCI, 5 mM MgCI2, 5% glycerol, 2.5 mM Hepes (pH 7.9), 1 pLg of polydeoxyinosinic-deoxycytidylic acid, and 20 jig of BSA for 30 min at 22°C. Polyclonal antipeptide antibodies to Spl and Egr-1 (Santa Cruz Biotechnology, Santa Cruz, CA) were incubated with nuclear extracts 15 min before the addition of the probe. 24. d77mEgr-CAT was constructed with the use of an oligonucleotide beanng the Oligo Bm (12) sequence as the 5' primer for the polymerase chain reaction. Transfections in BAECs were performed with 10 .Lg of reporter plasmid and the calcium phosphate protocol (10). The cells were incubated with PMA (100 ng/ml), cotransfected with CMV-Egr-1, or injured with a sterile comb (8), and then incubated for 36 hours at 37°C. CAT activity was assessed by the two-phase fluor diffusion technique (13) and was normalized to the amounts of protein in the cell lysate. 25. Recombinant Egr-1 was incubated with 32P-Oligo B (12) for 30 min at 22°C and applied to a running nondenaturing 5% polyacrylamide gel at the times indicated. Alternatively, a 1 000-fold molar excess of the unlabeled cognate was added after the 30-min incubation and applied to the gel (21). 26. Increasing amounts of recombinant Egr-1 were applied to a solution in which Spl was preincubated with 32P-Oligo B (12) for 30 min at 22°C (Fig. 3B, left side). Alternatively, 32p-Oligo B was incubated with a fixed concentration of Spl and decreasing amounts of Egr-1 (Fig. 3B, right side) (21). 27. Protein immunoblots were analyzed with polyclonal antibodies to Egr-1 and Spl (1:2500; Santa Cruz Biotechnology) and to PDGF-B (1:200; Genzyme). Immunoreactive proteins were detected by enhanced chemiluminescence (Amersham) with horseradish peroxidase-linked donkey secondary antiserum to rabbit immunoglobulin at 1 10,000 dilution. 28. J. T. Kadonaga, K. A. Jones, R. Tjian, Trends Biochem. Sci. 11, 755 (1986); K. A. Jones, J. T. Kadonaga, P. A. Luciw, R. Tjian, Science 232, 755 (1986). 29. B. Christy and D. Nathans, Proc. Natl. Acad. Sci. U.S.A. 86, 8737 (1989). 30. We thank F. J. Rauscher IlIl for recombinant Egr-1, V. P. Sukhatme for CMV-Egr-1, M. A. Frosch for critical review of the manuscript, and M. A. Gimbrone Jr. for enthusiastic support. Supported in part by grants from NIH (T.C.) and the American Heart Association (V.L.). L.M.K. is a C. J. Martin Postdoctoral Research Fellow (National Health and Medical Research Council of Australia) and a recipient of a J. William Fulbright Postdoctoral Research Award. T.C. is an Established Investigator of the American Heart Association.