G-CSF stimulation induces phosphorylation and cytosolic sequestration of Bad in ▵716 cells.

Ba/F3 cells transfected with the Δ716 G-CSFR were serum- and cytokine-deprived for 4 hours, then stimulated with 100 ng/mL G-CSF for the indicated times and lysed. (A) Whole-cell lysates were resolved by SDS-PAGE and transferred to nitrocellulose for blotting with mouse anti-Bad. Phosphorylated Bad is indicated by an upward shift in mobility (upper tick mark) compared with unphosphorylated Bad (lower tick mark). (B) Whole-cell lysates were immunoprecipitated with anti-Bad monoclonal antibody or (C) an irrelevant antibody (anti–G-CSFR) and blotted with a rabbit polyclonal antibody to 14-3-3.