Activation of MEK-1/2 during cell-cycle progression.

HL-60 cells were prepared for FACS analysis as described in “Materials and methods.” Graph A shows a representative control with unspecific mouse IgG antibodies. Similar results were obtained with rabbit and goat controls (data not shown). All cells were clearly positive when probed with ERK-2 or MEK-1 antibodies (graphs B,C), and only slightly positive when stained with phosphospecific antibodies against activated PP-ERK-1/2 and PP-MEK-1/2 (graphs D,E). Graph F shows the cell-cycle profile of untreated HL-60 cells stained with propidium iodide. Additionally, PP-MEK-1/2 staining revealed 2 strongly positive populations, which correlated with the G₀/G₁and G₂/M phases of the cell cycle (graphs E,F). Treatment of HL-60 cells with excess thymidine (2.5 mM) for 10 hours: G, staining with anti–PP-MEK-1/2 antibodies; H, cell-cycle profile. Treatment of HL-60 cells with 5 μg/mL vinblastine for 10 hours: I, staining with anti–PP-MEK-1/2 antibodies; J, cell-cycle profile.