Figure 4.

FAK and MLC-2 Phosphorylation and Rho activation are LRP-1 dependent in 3D matrix. Wild-type FTC-133 (treated with or without RAP) and clonal cells (shCTRL and shLRP-1) were seeded in 3D collagen matrix for 3 h and 24 h. Whole-cell extracts were subjected to Western-blot analysis to evaluate the level of FAK activation (A) and the phosphorylation of MLC-2 (B). The respective phosphorylation rates of FAK (A) and MLC-2 (B) were determined as intensity ratios of phospho-protein to corresponding pan-protein and expressed in relative units +/− SD, with a value of 100% ascribed to wild-type cells (right panels). GAPDH antibody was used as a loading control. (C) Cell lysates were incubated with GST-RBD beads. The bound RhoA was detected with a monoclonal anti-RhoA antibody (Top). The relative amount of total RhoA in the cell lysates were assessed by using a monoclonal antibody against RhoA (Bottom). Quantitative analysis of the GTP-RhoA associated with RBD beads was obtained by densitometry. The amount of RhoA bound to RBD was normalized to the RhoA content of cell extracts. *, P<0.05; ***, P < 0.001.