Figure 4

Effect of NO on HO-1 mRNA deadenylation and stability. A. NIH3T3 cells were treated as controls or with NO, and total RNA samples were assayed for HO-1 mRNA deadenylation. To compensate for the differences in the starting amount of HO-1 mRNA between control and NO-treated cells where HO-1 mRNA is induced immediately after the SPER/NO treatment, 35 μg and 6 μg of total mRNA were used in a cleavage reaction from untreated controls and NO-treated samples, respectively. Residual full-length HO-1 mRNA and the 3' end fragment carrying the poly(A) tail are indicated. FD represents HO-1 mRNA that was fully deadenylated in vitro. B. The upper boundary of the mRNA was set at a position below which 95–97% of the total HO-1 mRNA band area was included. The size of the poly(A) tail was estimated by comparing the migration of the upper bound to that of the FD (522-nt) and 623-nt size markers. C. A fraction of the samples was used without further modification to assess the half-life of HO-1 mRNA by northern blotting. Quantification in panels B and C shows the fitted decay lines calculated from two independent experiments. Data points show the mean and standard errors.