HIV vpr disruption of nuclear envelope

HIV vpr disruption of nuclear envelope - REPORTS 12. T....

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12. T. Pfeiffer, S. Schuster, S. Bonhoeffer, Science 292 , 504 (2001). 13 . C. V. Dang, G. L. Semenza, Trends Biochem. Sci. 24 , 68 (1999). 14. Construction and assay of the S6K1-GST construct were described previously ( 22 ). For the construction of the GST- D C-S6K1 and D3E,K100Q-S6K1-GST plas- mids, similar cloning strategies were used. 15. Total RNA was isolated from HEK293 cells as de- scribed ( 23 ). Deacylation of tRNA was accomplished by mild alkaline hydrolysis in 0.1 M tris-HCl (pH 8.0) at 75°C for 5 min. Total RNA was electrophoresed as described ( 23 ), and resolved tRNA was visualized with ethidium bromide staining before being trans- ferred to a nylon membrane by electroblotting. The individual tRNA species were probed with radiola- beled oligonucleotides as follows: for tRNALeu, 5 9 - GCGCCTTAGACCGCTCGGCCACG-3 9 ; for tRNAHis, 5 9 -GGTGCCGTGACTCGGATTCGAACCG-3 9 ; and for tRNAThr, 5 9 -GCGAGAATTGAACTCGCG-3 9 . 16. C. J. Lynch, H. L. Fox, T. C. Vary, L. S. Jefferson, S. R. Kimball, J. Cell. Biochem. 77 , 234 (2000). 17 . Transiently transfected, HA-tagged mTOR from HEK293 cells was extracted after a 16-hour serum starvation and immunoprecipitated with a monoclo- nal antibody to HA. The immunocomplex was washed once with 1 M NaCl in assay buffer [30 mM Mops (pH 7.5), 5 mM NaF, 20 mM b -glycerol phos- phate, 1 mM dithioerythritol, 0.1 % Triton X-100, and 10 % glycerol] and twice with assay buffer alone. 1 m g of a kinase-inactive, purified, soluble S6K1 substrate (S6K1-D3E,K100Q-GST ) or GST-4E-BP1 was added along with assay buffer containing 10 mM MgCl 2 and 1 mM ATP and incubated for 30 min at 30°C. Quantitation for K m measurements was carried out with scanning densitometry and ImageQuant software (Molecular Dynamics). 18. F. M. Gribble et al. , J. Biol. Chem. 275 , 30046 (2000). 19. A. M. Edelman, D. K. Blumenthal, E. G. Krebs, Annu. Rev. Biochem. 56 , 567 (1987). 20. T. Gaal, M. S. Bartlett, W. Ross, C. L. Turnbough Jr., R. L. Gourse, Science 278 , 2092 (1997). 21. M. Derenzini et al. , J. Pathol. 191 , 181 (2000). 22. N. Pullen et al. , Science 279 , 707 (1998). 23. J. A. Enriquez, G. Attardi, Methods Enzymol . 264 , 183 (1996). 24. Confluent cultures of HEK293 cells were treated as described ( 9 ), washed with PBS, and drained thor- oughly. The cells were then scraped into 500 m lo f water and sonicated with four 10-s pulses. Cell debris was removed by centrifugation, and sulfosalicylic acid was added to the supernatant to a final concen- tration of 2%. The samples were then placed on ice for 30 min followed by centrifugation to remove precipitated proteins. The extracts were then ana- lyzed for amino acid content with a Biochrom 20 plus amino acid analyzer (Amersham-Pharmacia Biotech).
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HIV vpr disruption of nuclear envelope - REPORTS 12. T....

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