Sustained activation of Akt in ▵716 cells in response to G-CSF.

Ba/F3 cells transfected with the WT or Δ716 G-CSFR were serum- and cytokine-deprived for 4 hours. The cells were stimulated with 100 ng/mL G-CSF for the indicated times, then lysed and immunoprecipitated with anti-Akt1. The immune complexes were washed and incubated in kinase buffer containing 500 μmol/L ATP, 75 mmol/L MgCl₂, 1 μCi of ³²PγATP, and 10 μg histone H2B for 20 minutes at 30°C. The reactions were subjected to SDS-PAGE. Phosphorylation of histone H2B was quantified with a PhosphorImager using ImageQuant software. (A) Akt activity was measured as the fold-increase in histone H2B phosphorylation over unstimulated samples. Inset is a linear representation of the data. (B) Detection of histone H2B phosphorylation from samples in (A).