Ser585 of βc and PI 3-kinase activity are required for GM-CSF–mediated cell survival. (A) Cells were washed and plated out at 5 × 105 cells/mL in medium containing either no factor (white bars), 2 ng/mL GM-CSF (hatched bars), 20 ng/mL GM-CSF (dark gray bars), or 20 ng/mL control cytokine, IL-2 (black bars). After 48 hours, cells were costained with annexin V and propidium iodide and analyzed by flow cytometry as described in “Materials and methods.” The average of duplicate samples ± the standard deviations (SDs) are indicated. (B) CTL-EN cells expressing the wt GM-CSF receptor were washed and plated out as above in medium containing either no factor (solid) or 50 ng/mL GM-CSF (open) with increasing amounts of pharmacologic inhibitor. The SB203580 p38 MAP kinase inhibitor was used at 0, 0.2, 0.6, and 1.2 μM. The PD98059 MEK inhibitor was used at 0, 6, 12, and 20 μM. The LY294002 PI 3-kinase inhibitor was used at 0, 10, 30, and 50 μM. The AG490 JAK2 inhibitor was used at 0, 5, 20, and 30 μM. After 48 hours, cell viability was examined by the MTS reduction assay and measured at 490 nm. The average of triplicate samples ± SD are plotted.
