Figure 2. Bufalin reduces Axl protein level and suppresses promoter activity.

(A) Cells (3 × 10⁵ cells/ 60 mm dish) were treated with 20, 40, or 80 nM bufalin for 24 h. Axl protein levels were determined by Western blot analysis. (B) For RT-PCR, total RNAs extracted from the cells treated with the indicated concentrations of bufalin for 8 h were used to measure Axl mRNA levels. (C) To examine the effect of bufalin on Axl promoter activity, H460/pGL3-Axl cells (3 × 10⁴ cells) were incubated with 20, 40, or 80 nM bufalin for 4 or 8 h, and the luciferase activities were then measured (**P<0.01, ***P<0.001 (4 h), ^###P < 0.001 (8 h), vs untreated group). (D) H460/pGL3-Axl cells were also treated with 40 nM bufalin for 0.5, 1, 2, 4, or 8 h, and then the luciferase activities were measured (*P<0.05, **P<0.01, ***P<0.001, vs untreated group).