Figure 5
From: The RhoA dependent anti-metastatic function of RKIP in breast cancer

RKIP activates RhoA through GEF-H1. (a) Representative western blots of VAV2 GEF activity pull-down assays with lysates prepared from BT20 cells. Numbers are shown for quantified active VAV2 bands normalized with total VAV2 protein. (b) (Left panel) Representative western blots of lysates prepared from control knockdown (shCTR), or five different VAV2 knockdowns (shVAV22.1–2.5) BT20 cells. (Middle panel) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of BT20 cells shown in the left top panel. (Right panel) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. (c) (Left panel) Representative western blots of lysates prepared from control knockdown (shCTR), or two different GEF-H1 knockdowns (shGEF-H12.2–2.3) BT20 cells. (Middle panel) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of BT20 cells shown in the left top panel. (Right panel) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. (d) (Top left panel) Representative images of immunofluorescent staining with E-Cad Ab and DRAQ5 of BT20 cells with different combinations of control knockdown, GEF-H1 knockdown (shGEF-H1), empty vector control (EVC) or RKIP expression. (Bottom left panel) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. (Right panel) Representative western blots of lysates prepared from the same set of cells shown in the left panel. (e) (Left panel) Representative results of invasion assays with same set of cells used in (d) showing number of invaded cells through Matrigel in each indicated BT20 cell lines (mean ± SE). (Right panel) Representative images of the stained invaded cells in indicated cell lines shown at the left panel. Unpaired Student's t-test (two-tailed) was used for all analyses with p < 0.05 considered significant. ns not significant.