FIG. 4.

IL-3 induces CREB phosphorylation and survival function partly via the PKA pathway. (A) IL-3 induces PKA-dependent CREB Ser¹³³ phosphorylation. Cytokine-starved Ba/F3 cells were pretreated with dimethyl sulfoxide (lanes 1 and 2) or PKI (lanes 3 and 4) for 30 min before being restimulated with IL-3 (IL3). After 5 min of stimulation, cells were harvested and analyzed by Western blot analysis with antibodies specific to phosphorylated CREB at Ser¹³³ (-CREB) and to all forms of CREB (CREB). The star indicates the position of the phosphorylated ATF-2. (B) Increase of survival of Ba/F3 cells by cAMP-elevating agents. Viable cell number was measured by trypan blue staining after 18 h of incubation in cytokine-free medium without (−) or with (+) forskolin (FsK 100 μM) plus IBMX. Numbers of viable cells are presented as percentages of the number of cells initially seeded. Results shown are means ± standard deviations from two independent experiments performed in duplicate. (C) Decrease of cell viability by PKI. Ba/F3 cells were cultured in IL-3-containing medium supplemented with various doses of Myr-PKI (lanes 2 and 3, 10 and 20 μM). Cells cultured in cytokine-free medium were included as a negative control (lane 4). Numbers of viable cells under various treatments were determined and are presented as a percentage of the number of cells initially seeded. Shown are representative results from three independent experiments performed in duplicate.