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. 2006 Jun 6;34(10):3044–3056. doi: 10.1093/nar/gkl386

Figure 4.

Figure 4

NO• stabilizes CURE-containing mRNA but inhibits its translation through an Erk1/2-dependent mechanism. (A) Effect of the Erk1/2 inhibitor, PD98059 (PD; 30 µM), on LUC mRNA levels and LUC activity, respectively. THP-1 cells, transfected with pGL3/CURE, mutant pGL3/CUREmut or control pGL3, were treated with ActD (2.5 µg/ml) for 30 min (for mRNA determinations only) and then incubated with GSH (400 µM) or GSNO (400 µM) for 5 h to measure LUC mRNA by TaqMan® RT–PCR or for 20 h to measure LUC activity. (B) Effect of a Mek1 dominant-negative mutant on LUC mRNA levels and LUC activity, respectively. THP-1 cells, co-transfected with pGL3/CURE or mutant pGL3/CUREmut or control pGL3 plus either pUSEamp (empty vector) or pMEK1-DN (dominant-negative Mek1), were similarly treated as in A for measurement of LUC mRNA levels and LUC activity. Data, presented as percentage relative to LUC mRNA level or LUC activity of pGL3, are the mean ± SEM of three to six independent experiments. (C) Effect of NO• on the expression of MAP3K7IP2, a naturally-occurring, CURE-containing gene. THP-1 cells (1 × 107) were pretreated with SB (0.1 µM) or PD (30 µM) for 30 min. After 20 h incubation of GSH (400 µM) or GSNO (400 µM), cells were then lysed for western blotting. Each experiment was repeated twice with similar results.