FIG. 5.

Effect of TNF-α/Cholix on MAP kinases in HepG2 cells. A, Time course of MAP kinase phosphorylation induced by TNF-α and Cholix. HepG2 cells (2 × 10⁵ cells/well) in 48-well plates were incubated with 10 μg/ml wild-type Cholix (WT) or mutant Cholix(E581A) (MT) in the presence or absence of TNF-α (25 ng/ml) for the indicated times. Then, cells were lysed with 1×SDS sample buffer for immunoblotting with the indicated antibodies. Experiments were repeated three times with similar results. B, Effect of MAPK inhibitors on MAPK phosphorylation and apoptotic signals induced by TNF-α/Cholix. HepG2 cells were treated for 30 min with 10 μM SP600125 (SP), 10 μM U0126 or 20 μM SB20350 (SB), followed by incubation for 12 h with 10 μg/ml wild-type Cholix (WT) or mutant Cholix (MT) in the presence or absence of TNF-α (25 ng/ml). Then, cells were lysed with 1×SDS sample buffer for immunoblotting with the indicated antibodies. Experiments were repeated three times with similar results.