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. 2000 Feb 29;97(6):2491–2496. doi: 10.1073/pnas.050586597

Figure 3.

Figure 3

Inhibition of Ace1 binding to CUP1 promoter DNA by DEA/NO. Electrophoretic mobility-shift assays were performed using 6% polyacrylamide gel. (A) Cell extracts (3 μg of protein/lane) from KKY7 (lane 2) or 217ACE (lanes 3, 4, and 5) were incubated with 0.5 mM KCN (lane 3) or 0.5 mM BCS (lane 4) at room temperature for 10 min, followed by incubation with 32P-labeled CUP1 UASc DNA (60,000 dpm) and 0.1 mM CuI(CH3CN)2ClO4 (lanes 1, 2, and 5) or 0.1 M acetonitrile (lane 3 and 4) for 10 min at room temperature. Lane 1 contains no cell extract. (B) Cell extracts (217ACE) were incubated with the indicated concentration of DEA/NO (lanes 1–5) or 4 mM decomposed DEA/NO (lane 6) for 10 min at room temperature. To the mixtures were then added 32P-labeled CUP1 UASc DNA (60,000 dpm) and 0.1 mM CuI(CH3CN)2ClO4, and they incubated for 10 min at room temperature. Electrophoresis and exposure to x-ray film are described in Experimental Procedures. Representative experiments are shown.