Activation of ERK by Triphala. A) Cells were treated with varying concentrations of Triphala for 24 h or with 60 μg/ml Triphala for different time intervals in a time-dependent experiment. Expression and phosphorylation of ERK (Thr-202/Tyr-204) and MEK-1 (Ser-217/221) was determined by western blotting. B) The effect of Triphala on the kinase activity of ERK was determined using a kit from Cell Signaling Technology, measuring the phosphorylation of Elk-1 at Ser-383. C, D) Effect of ERK inhibitor on Triphala induced apoptosis and activation of p53. Cells were pretreated with 20 μM U0126, MEK-1 inhibitor for 1 hour followed by exposure to 60 μg/ml Triphala for 4 h in the presence of inhibitor. Apoptosis and p53 was determined by western blot or using cell death detection kit. E) Effect of pifithrin and U0126 on the transcriptional activation of p53. These assays were performed 2–3 times independently with similar results. Statistical significance was determined by one-way ANOVA followed by Bonferroni's post hoc analysis for multiple comparisons. *Significantly different compared with PBS-treated control (P < 0.05). **Significantly different compared with Triphala treatment (P < 0.05).