FIGURE 4.

MAPK signaling mediates Sam68 phosphorylation and migration in MDA-MB-231 breast cancer cells. A, HaCaT and MDA-MB-231 cells were starved for 18 h and treated with either vehicle or 50 ng/ml HGF for 4 h. CD44v5 expression was determined with specific antibodies. Total p38 served as a loading control. B, MDA-MB-231 cells were starved for 18 h, pretreated with either vehicle or 10 μm MEK1/2 inhibitor (U0126) for 30 min, and subsequently treated with 50 ng/ml HGF for 30 min. Sam68 was immunoprecipitated (IP) from whole cell lysates and then subjected to Western blotting (IB) using Sam68-specific antibodies. Normal rabbit IgG served as a specificity control. Upshifted Sam68 band indicates multisite phosphorylation. C, Boyden chamber migration assays preformed with MDA-MB-231 stimulated with HGF in the presence or absence of MEK1/2 inhibitor, U0126. Inset shows HGF-induced ERK1/2 activation; ERK1/2 protein levels served as loading control. * denotes significance (p < 0.05) relative to vehicle and between indicated groups, as determined by unpaired Student's t test.