Subcellular localization and physical association of LIMK1 with MT1-MMP. A) Immunofluorescence analysis of LIMKT508EE and MT1-MMP in BPHLCA (a-c) and PC3 (d-f) cells. a-c: Co-localization of MT1-MMP (red) and LIMK1 (green)(merged image) in these cells was mainly to the perinuclear regions at the ER/Golgi area. Colocalization of MT1-MMP with LIMK1 was also at the plasma membranes (yellow arrows). These cells showed intense staining and accumulation of LIMK1 in the ruffling membranes (Insert: yellow arrows). Scale bar 5 μm. d-f: Colocalization of LIMK1 and MT1-MMP in PC3 cells. Immunofluorescence analysis of MT1-MMP and LIMK1 showed strong staining of both proteins in the Golgi areas and in transport vesicles (yellow arrows). Scale bar 5 μm. B) Analysis of colocalization using overlap coefficient of actual pixels in designated areas (red circles) at the Golgi region and at the membrane for both BPHLCA and PC3 cells. C) Coimmunoprecipitation (Co-IP) and D: Reverse Co-IP of MT1-MMP and LIMK1 using anti-LIMK1 (mouse monoclonal) and anti-MT1-MMP (rabbit polyclonal) antibodies and PC3 cell extracts (500 μg) showing pull down of MT1-MMP and LIMK1 in immunoprecipitates. Nsp mAb: Nonspecific mouse monoclonal antibodies. Rabbit serum and nonspecific mouse mAb were used as the negative controls.