Figure 3. An inhibitory effect of silibinin on the phosphorylation of ERK1/2. (A) Cells were treated with the indicated dose of silibinin (0, 25, 50, 75, or 100 µM) for 24 hrs, and then cell lysates were subjected to SDS-PAGE followed by Western blotting and immuno-probing with anti-phospho-ERK1/2, anti-phospho-p38, anti-phospho-JNK1/2, or anti-phospho-Akt antibodies. Protein reactivity was visualized with an ECL detection system. For determination of the effects of MEK inhibitor (U0126) and silibinin on the activity of MMP-2 and u-PA and cell invasion, cells were plated in culture dishes and pre-treated with U0126 (10 or 20µM) for 30 min, and then incubated in the presence or absence of silibinin (25 µM) for 24 hrs.Afterward, the culture media were subjected to gelatin and casein zymography to analyze the activities of MMP-2 (B) and u-PA (C), respectively, and cells were subjected to analyses for invasion (D) as described in MATERIALS & METHODS. Determined activities of these proteins were subsequently quantified by densitometric analysis, with control being 100% as shown below the gel data. Nuclear extracts were subjected to SDS-PAGE followed by Western blotting and immunoprobing with anti-NF-B, c-Jun, c-Fos, or C23 antibodies (E) as described in MATERIALS & METHODS. Data represent the mean ± SD of at least 3 independent experiments. (*P < 0.05; **P < 0.01).