Figure 3.

Elevation of cAMP affects the rate of Kv1.1 RNA degradation with only a small affect on transcription of Kv1.1. (A) Kv1.1 RNA half-life was determined by initiating pharmacological treatments on duplicate cultures/treatment/time point and harvesting RNA at the indicated times. RPA analysis was used to follow Kv1.1 RNA levels relative to cyclophilin. Cyclophilin levels did not change significantly with any of these treatments at any of these time points. Error bars are not shown because error was ≤2% at each time point. Half-life measurements were made by plotting results on a semi-log plot (50% level indicated by the thin horizontal line.) Three independent experiments, with n = 2 for each treatment at each time point, were conducted with the same results. (B) For nuclear run-on transcription assays, nuclei were prepared from cells treated for 6 h with vehicle (control) or Iso/IBMX. Quantitation represents the average of the ratio of Iso/IBMX over control for the antisense strand with the number of measurements indicated above the bar. Standard deviation for Kv1.1 = 0.23 and for LDH = 1.82. No change would be equal to 1.0 as indicated by the horizontal line on the graph. The radiographic image is one representative experiment. The (+) indicates the sense strand probe, and the (−) indicates the antisense strand probe.