An fMRI Investigation of Emotional Engagement in Moral Judgment

نویسندگان

  • D. Ferring
  • R. P. Jansen
چکیده

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The yeast ORFs were cloned using the strategy described previously (5), except every step was done in a 96-well format. Plasmid DNAs confirmed by DNA sequencing were reintroduced into both yeast (Y258) and E. colt (DH5a). The library contains 5800 unique ORFs. 10. For details of 96-well format protein purification protocol, a full list of results from all the experiments, and the design of the positive identification algorithms, please visit our public Web site (http:// bioinfo.mbb.yale.edu/proteinchip) and supplementary material at Science Online (www.sciencemag.org/ cgi/content/full/1062191/DC1). 11. Biotinylated calmodulin (CalBiochem, USA) was added to the proteome chip at 0.02 pIg/pl in phosphatebuffered saline (PBS) with 0.1 mM calcium and incubated in a humidity chamber for 1 hour at room temperature. Calcium (0.1 mM) was present in buffers in all subsequent steps. The chip was washed three times with PBS at room temperature (RT, 25?C). Cy3-conjugated streptavidin (Jackson IR, USA) (1:5000 dilution) was added to the chip and incubated for 30 min at RT. After extensive washing, the chip was spun dry and scanned using a microarray scanner; the data was subsequently acquired with the GenePix array densitometry software (Axon, USA). 12. S. S. Hook, A. R. Means, Annu. Rev. Pharmacol. Toxicol. 41, 471 (2001). 13. M. S. Cyert, R. Kunisawa, D. Kaim, J. Thorner, Proc. Natl. Acad. Sci. U.S.A. 88, 7376 (1991). 14. D. A. Stirling, K. A. Welch, M. J. Stark, EMBO J. 13, 4329 (1994). 15. F. Bohl, C. Kruse, A. Frank, D. Ferring, R. P. Jansen, EMBOJ. 19, 5514 (2000); E. Bertrand et al., Mol. Cell 2, 437 (1998). 16. D. C. Winter, E. Y. Choe, R. Li, Proc. Natl. Acad. Sci. U.S.A. 96, 7288 (1999). 17. C. Schaerer-Brodbeck, H. Riezman, Mol. Biol. Cell 11, 1113 (2000). 18. K. Homma, J. Saito, R. Ikebe, M. Ikebe, J. Biol. Chem. 275, 34766 (2000). 19. J. Menendez, J. Delgado, C. Gancedo, Yeast 14, 647 (1998). 20. G. Odorizzi, M. Babst, S. D. Emr, Trends Biochem. Sci. 25, 229 (2000); D. A. Fruman et al., Annu. Rev. Biochem. 67, 481 (1998); T. F. Martin, Annu. Rev. Cell Dev. Biol. 14, 231 (2000); S. Wera, J. C. T. Bergsma, FEMS Yeast Res. 1, 1406 (2001). 21. Liposomes were prepared using standard methods (30). Briefly, appropriate amounts of each lipid in chloroform were mixed and dried under nitrogen. The lipid mixture was resuspended in TBS buffer by vortexing. The liposomes were created by sonication. To probe the proteome chips, 60 ,il of the different liposomes were added onto different chips. The chips were incubated in a humidity chamber for 1 hour at RT. After washing with TBS buffer for three times, Cy3-conjugated streptavidin (1:5000 dilution) was added to the chip and incubated for 30 min at RT. 22. Positives were identified using a combination of the GenePix software which computes a local intensity background for each spot and a series of algorithms we developed. Details can be found at http://bioinfo.mbb.yale.edu/proteinchip and at www.sciencemag.org/cgi/content/full/1062191/ DC1. 23. M. C. Costanzo et al., Nucleic Acids Res. 29, 75 (2001). 24. M. Gerstein, Proteins 33, 518 (1998). 25. K. Ansari et al., J. Biol. Chem. 274, 30052 (1999). 26. Y. Barral, M. Parra, S. Bidlingmaier, M. Snyder, Genes Dev. 13, 176 (1999). 27. Y. Li, T. Kane, C. Tipper, P. Spatrick, D. D. Jenness, Mol. Cell. Biol. 19, 3588 (1999). 28. I. Arnold et al., J. Biol. Chem. 274, 36 (1999). 29. S. Chu et al., Science 282, 699 (1998). 30. A. Casamayor et al., Curr. Biol. 9, 186 (1999); R. Guerra, M. L. Bianconi, Biosci. Rep. 20, 41 (2000). 31. M. Pardo et al., Yeast 15, 459 (1999). 32. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, lie; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gin; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. X indicates any residue. 33. We thank K. Nelson and S. Dellaporta for providing invaluable help. We also thank A. Kumar, G. Michaud, and C. Costigan for providing comments on the manuscript. This research is supported by grants from NIH. H.Z., A.C., and R.J. were supported by postdoctoral fellowships from the Damon Runyon-Walter Winchell Foundation, the Spanish Ministerio de Ciencia y Tecnologia, and by an IBM Graduate Research Fellowship, respectively.

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تاریخ انتشار 2014