Ser585 of β_c is important for NF-κB activation in response to GM-CSF. (A) Cells were electroporated (10⁷ cells/electroporation) with 20 μg Ig-κB-firefly luciferase reporter plasmid (pTK81-IgK) and 1 μg Renilla luciferase control vector (pRL). After 24 hours of culture, the cells were plated in medium containing either no factor (nil), 20 ng/mL GM-CSF, or 20 ng/mL IL-2 for a further 12 hours. Cell extracts were then made and luciferase activity was measured using the dual-luciferase assay system. Triplicate samples ± SD were assayed. To examine the ability of GM-CSF to regulate IκB phosphorylation (B), cells were factor deprived for 18 hours in medium containing 0.5% FCS and then stimulated with 50 ng/mL GM-CSF for 0, 5, 15, and 30 minutes. Following stimulation, cells were lysed and lysates were subjected to SDS-PAGE and immunoblotted sequentially with anti–phospho-IκB (B, top panel) and anti-MAP2 (B, bottom panel) antibodies to demonstrate equal loading. The ability of 2 independent inhibitors of NF-κB to block GM-CSF–mediated survival was also examined (C). CTL-EN cells expressing the wt GM-CSF receptor were washed and plated out at 4 × 10⁵ cells/mL in medium containing either no factor (open bar) or 50 ng/mL GM-CSF (solid bar). The peptide inhibitor of NF-κB translocation, SN50, and a control peptide, SN50M, were used at 0, 2, and 20 μM. The inhibitors of IκB phosphorylation, BAY 11-7082 and BAY 11-7085, were used at 0, 0.6, 1.2, and 2 μM. After 48 hours, cell viability was examined by the MTS reduction assay at 490 nm. Triplicate samples ± SD were plotted.