LIMK1 expression positively regulates surface localization of MT1-MMP in BPH and PC3 cells. A) Flow cytometric analysis of MT1-MMP cell surface expression in transfected BPH cell lines. Two-parameter histogram of the surface staining of MT1-MMP with fluorescence intensities in the X-axis and number of cells in the Y-axis in BPH cells expressing LIMK1. Black histogram represents unstained cells. Red histogram represents population of fluorescent cells within each sample. B) Quantitative analysis of fluorescent cells. For densitometric analysis, the BPHLCA cells that were emitting fluorescence were gated out from the nonfluorescent cells and calculated as the percentage of the fluorescent control (BPHV) cells. Data represents the mean ± SD of three independent experiments. *P = 0.0005. C) Surface biotinylation of MT1-MMP following knock down of LIMK1 in PC3 cells. Immunoblot of biotinylated MT1-MMP in the streptavidin bead bound MT1-MMP. Lane 1 non-biotinylated cells, Lane 2: control shRNA expressing cells, Lane 3: LIMK1 shRNA expressing cells. D) Immunoblot analysis of LIMK1 in PC3 cells expressing control shRNA or LIMK1 shRNA used for surface biotinylation assays. Data show representative images of three independent experiments.