Enhanced activation of MAPK and Akt signaling by pretreatment of MO7e cells with SDF-1α.

Factor-starved MO7e cells were preincubated with or without 100 ng/mL of either SDF-1α, MIP-1α, or RANTES for 30 minutes or the indicated periods of time (Aiii), followed by treatment with 10 ng/mL SLF or 1 ng/mL GM-CSF for 5 minutes. In some experiments, cells were pretreated with PD98059 for 1 hour before SLF treatment (Aiv). (A) ERK immunoprecipitates obtained from total cell lysate using anti–phospho-ERK mAb were subjected to MAPK activity assay as described in “Materials and methods.” Recombinant active MAPK (20 ng) was included as a positive control (Ai). These results are representative of 3 independent experiments. (B) Phosphorylation levels of Akt were determined by Western blotting with phosphospecific antibodies to Akt (Ser473). The amount of total Akt is shown as a loading control in the bottom panels of B. This is a representative of 3 separate experiments with similar results.