Dtt

Fig. 4. Redox sensitivity of RNA-protein binding assay. RNA-protein gel shift assay of the 177-nucleotide Topo II3' UTR riboprobe and HeLa cellular protein extracts pretreated with thiol-reducing or -oxidizing agents. (A) Protein extracts from asynchronously growing HeLa cells were prepared with extraction buffer without any DTT. RNA-protein binding reactions were carried out with protein extracts that were pretreated with (lane 2) or without (lane 1) 2 mM DTT. Lanes 4-9, protein extracts from asynchronously growing HeLa cells were prepared with regular extraction buffer containing 2 mM DTT and RNA-protein binding reactions were carried out in the presence of increasing concentrations of diamide (0.1,0.5,1,5, and 10 mA/). Lane 3 represents a control reaction without any protein extract.

In Vivo Manipulation of Redox State and in Vitro RNA-Protein Binding Assay

Asynchronously growing HeLa cells are treated with 20 mM /V-acetyl-L-cysteine (NAC) and assayed for NAC uptake, reduced and oxidized glutathione content, RNA-protein binding, and Topo II mRNA levels. The pH of the NAC is adjusted to pH 7.0 with sodium bicarbonate. Intracellular reduced and oxidized glutathione as well as NAC levels are assayed according to previously published protocols.18'19 Cell pellets are homogenized in 50 mM potassium phosphate buffer (pH 7.8) containing 1.34 mMdiethylenetriaminepentaacetic acid. Total glutathione content is determined in sulfosalicylic acid [5% (w/v) SSA] extracts by the method

18 R. V. Blackburn, D. R. Spitz, X. Liu, S. S. Galoforo, J. E. Sim, L. A. Ridnour, J. C. Chen, B. H. Davis, P. M. Corry, and Y. J. Lee, Free Radic. Biol. Med. 26,419 (1999).

19 L. A. Ridnour, R. A. Winters, N. Ercal, and D. R. Spitz, Methods Enzymol. 299, 258 (1999).

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