P5N-I of primitive day-5 RBCs. (A) The mRNA expression of P5N-I was determined by Northern blot analysis of 10 μg RNA obtained from RBCs before (t0) and after a 4-hour incubation with 1 μM NE and 10 μM propranolol (P). The expression level was compared with the expression level in day-11 RBCs. Shown is 1 of 3 independent experiments. (B) P5N-I enzyme activity during a 12-hour incubation with or without 10 μM NE. Shown are the mean values and SDs of 3 independent experiments. The mean enzyme activities of an incubation with 10 μM NE of day-11 RBCs, taken from Figure 4E, were added. (C) Western blot analysis for the phosphorylation of CREB-like proteins in RBCs of day 5, compared with RBCs of day 11. The RBCs were incubated for one hour in the presence of 1 μM NE ± 10 μM propranolol (P). Equal amounts of cells (1 × 106 per lane) were analyzed with specific antibodies (see Figure 5 for details). Shown is 1 of 3 experiments.
