Trib1 enhances phosphorylation of MAPK. (A) Phosphorylation of ERK1/2 on stimulation of HeLa cells by PMA. Overexpression of Trib1 enhances PMA-induced ERK1/2 phosphorylation. (B) Trib1 induces extensive phosphorylation by IL-3 stimulation. IL-3 was added after 16 hours starvation and phosphorylated ERK was examined in Trib1-positive (21) and -negative (35) Hoxa9/Meis1-AMLs (left) and primary bone marrow cells transduced with Trib1 (T), Tirb1/Hoxa9-Meis1 (T + H/M) retroviruses, or only Hoxa9-Meis1 (H/M) retroviruses. ERK phosphorylation in BaF3 cells with same treatment is shown as control. Expression of Hoxa9, Meis1, and Trib1 proteins in leukemia cells are shown by Western blotting. Each box represents a separate experiment. (C) ERK is constitutively phosphorylated in IL-3 depletion. Phosphorylation of ERK was examined after IL-3 removal from growth medium for the same AMLs and primary bone marrow cells used in panel B. Durations of IL-3 starvation are indicated. Each box represents a separate experiment. Three groups of lanes in the same filter were cut and pasted together, and the borders were indicated by vertical lines. (D) Trib1 expression suppressed apoptosis during IL-3 starvation. Frequencies of annexin V–positive cells were compared between Trib1-positive (21) and -negative (35) AMLs 16 hours after IL-3 removal. The means ± standard deviations from 3 independent experiments are shown. *P < .05.