Matson et al_Science_1998

Matson et al_Science_1998 - 17. 18. 19. 20. 21. S. J....

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S. J. Leevers, H. F. Paterson, C. J. Marshall, Nature 369 , 411 (1994). 17. R. Marais, Y. Light, H. F. Paterson, C. J. Marshall, EMBO J. 14 , 3136 (1995). 18. Z. Lou, B. Diaz, M. S. Marshall, J. Avruch, Mol. Cell. Biol. 17 , 46 (1997). 19. H. Mischak et al. , ibid. 16 , 5409 (1996). 20. R. Finney and D. Herrera, Methods Enzymol. 255 , 310 (1995); B. Hallberg, S. I. Rayter, J. Downward, J. Biol. Chem. 269 , 3913 (1994). 21. L. Van Aelst, M. Barr, S. Marcus, A. Polverino, M. Wigler, Proc. Natl. Acad. Sci. U.S.A. 90 , 6213 (1993); S. A. Moodie, B. M. Willumsen, M. J. Weber, A. Wolfman, Science 260 , 1658 (1993); A. B. Vojtek, S. M. Hollenberg, J. A. Cooper, Cell 74 , 205 (1993); P. H. Warne, P. R. Viciana, J. Downward, Nature 364 , 352 (1993); H. Koide, T. Satoh, M. Nakafuku, Y. Kaziro, Proc. Natl. Acad. Sci. U.S.A. 90 , 8683 (1993). 22. S. J. Taylor and D. Shalloway, Curr. Biol. 6 , 1621 (1996). 23. J. de Rooij and J. Bos, Oncogene 14 , 623 (1997). 24. R. Marais, Y. Light, H. F. Paterson, C. S. Mason, C. J. Marshall, J. Biol. Chem. 272 , 4378 (1997). 25. All extraction procedures were done at 4°C. Cells were washed twice with phosphate-buffered saline, and each 10-cm dish of confluent cells was extract- ed in 300 m l of dilution buffer ( 17 ) containing 100 mM KCl and 5 mM MgCl 2 , but only 0.05% v/v 2-mercap- toethanol (medium-salt buffer). DNA was sheared, and extracts were clarified by centrifugation (13,000 g for 2 min); protein concentrations were measured with a Bio-Rad protein assay kit. Immu- noblots for endogenous Ras or Raf-1 were done with monoclonal antibodies (products no. 02120 and no. R19120, Transduction Laboratories). For Ras-Raf complex analysis by immunoblot, endogenous Ras or Raf-1 was immunoprecipitated from ; 15 mg of cellular protein with either 10 m g of Y13-238 or 10 m g of Raf-1 monoclonal antibody and probed by immu- noblotting for Raf-1 or Ras. For assay of Ras-asso- ciated GSTMek-1 activation, Ras was immunopre- cipitated from ; 3.5 mg of cellular protein with Y13- 238 (10 m g), washed four times with low-salt buffer, and assayed as described ( 17, 24 ). Ras immunopre- cipitates were eluted with extraction buffer (40 m l), diluted with dilution buffer (160 m l) ( 17 ), and repre- cipitated with Raf-1 monoclonal antibody (2 m g). Measurements of Ras-GTP were done as described ( 23 ). The cells were extracted in medium-salt buffer, and proteins ( ; 3 mg) were absorbed to bacterially expressed GSTRBD. Ras proteins were revealed by immunoblotting. In control experiments using GSTR89LRBD, Ras-GTP was not detected ( 13 ). 28 August 1997; accepted 17 February 1998 Integration of Environmental, Agronomic, and Economic Aspects of Fertilizer Management Pamela A. Matson,* Rosamond Naylor, Ivan Ortiz-Monasterio Nitrogen fertilization is a substantial source of nitrogen-containing trace gases that have both regional and global consequences. In the intensive wheat systems of Mexico, typical fertilization practices lead to extremely high fluxes of nitrous oxide (N
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Matson et al_Science_1998 - 17. 18. 19. 20. 21. S. J....

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