Sustained Erk activation in survival-inducing condition. (A) Phosphorylation of MAPKs upon anti-CD8 stimulation. FcRγ-deficient BMMCs expressing CD8/γ were incubated without IL-3 for 2 hours at 37°C followed by treatment with 0.03 μg/mL or 3 μg/mL anti-CD8 for the indicated periods. Total cell lysates were blotted with Abs for phosho-Erk (anti-pErk), phospho-JNK (anti-pJNK), phosho-p38 (anti-pp38), phosho-Akt (anti-pAkt), and Erk (Erk). Lysates were also precipitated with protein G–Sepharose, and the amount of anti-CD8–bound CD8/γ was determined by immunoblot with anti–FcRγ (bottom panel). (B) Erk activation is sustained for days. A total of 2 × 105 CD8/γ-bearing BMMCs were cultured in the presence of the indicated amounts of anti-CD8. Cells were harvested at day 0 (immediately after IL-3 depletion) and day 3. Total lysates were blotted with anti-pErk and anti-Erk Abs. Viability of each cell is shown below. (C) Effect of active MEK on BMMC survival. Mature BMMCs from normal B6 mice were infected with pMX-IRES-GFP vector alone (Mock; ○), wild-type MEK (MEK.WT; •), or constitutive active MEK (MEK.ΔSESE; ▵) with 100 ng/mL stem cell factor (SCF) and 10 ng/mL IL-3. Forty-eight hours after infection, cells were cultured in cytokine-free medium for 5 days. Viable GFP-positive cells were determined by flow cytometry and expressed as percentage of day 0. Data were means ± SDs of triplicate assays. Similar results were obtained from 5 independent experiments.
