Figure 1

HNK treatment down-regulates HGF-induced c-Met phosphorylation; and it inhibits both HGF- and CNI-mediated Ras activation and HO-1 over-expression. (A) 786-O cells were pre-treated with either HNK (20 μM)/vehicle alone for 2 h, and then treated with either HGF (50 ng/ml) or vehicle alone for 30 min. Following treatment, the cell lysates were used to measure the levels of phospho-c-Met, c-Met, and β-actin (internal control) by Western blot analysis. (B) 786-O cells were treated as described in (A). Following treatment, cell lysates were prepared utilizing a Ras activation kit as described under “Materials and Methods” section, and the expression of GTP-bound Ras was subsequently analyzed by Western blot. (C). 786-O cells were pre-treated with either CsA (5 μM)/vehicle alone for 2 h, and then treated with either HGF (50 ng/ml) or vehicle alone for 30 min. Following treatment, the expression of GTP-bound Ras was analyzed by Western blot. (D) 786-O cells were treated with either CsA (5 μM) or vehicle alone for 30 min and cell lysates were utilized to measure the levels of phospho-c-Met, c-Met, and β-actin by Western blot analysis. (E) 786-O cells were pre-treated with either HNK (20 μM) or vehicle alone for 2 h and then treated with different combinations of HGF (50 ng/ml), CsA (5 μM) or vehicle alone for 24 h. Following treatment, the cell lysates were used to measure the expression of HO-1 and β-actin. F. 786-0 cells were treated as described in (E), and following treatment nuclear and cytoplasmic fractions were isolated from cells, and Western blot analysis was performed to measure the expression of Nrf-2 in each of the fractions. The purities of nuclear and cytoplasmic fractions were evaluated by the expression of SP-1 and GAPDH respectively. (A to F) are representative of three independent experiments. For all Western blots (A–F), the bands from adjacent lanes were cropped from the same blot (full-length blots are included in a supplementary file).