The membrane fraction of B16BL6 cells was extracted using the ProteoExtract Native Membrane Protein Extraction Kit (Calbiochem). A 40-μg protein aliquot of each extract was fractionated by electrophoresis in a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) selleck chemicals membrane (Amersham, Arlington Heights, IL, USA). The membranes were blocked with a solution containing 3% skim milk, and then incubated overnight at 4°C with each of the following antibodies: anti-phospho-LIMK antibody, anti-LIMK antibody, anti-phospho-MLC antibody (Cell Signaling Technology, Beverly, MA, USA), anti-MMP-14

antibody (Calbiochem), anti-α2 integrin antibody (Chemicon Int. Inc., California, USA), anti-α4 integrin antibody (SantaCruz Biotechnology, CA, USA), anti-α5 integrin antibody (SantaCruz Biotechnology), and anti-Rho antibody (Upstate Biology, Charlottesville, VA, USA). Subsequently, the membranes were incubated for 1 h at room temperature with anti-rabbit IgG sheep

antibody coupled to horseradish peroxidase (Amersham). Reactive proteins were visualized using a chemiluminescence kit (Amersham) according to the manufacturer’s instructions. Mouse BAY 80-6946 research buy anti-β-actin monoclonal antibody (Sigma) was used as the primary antibody (internal

standard) for detecting β-actin protein. Reverse transcription-polymerase chain reaction Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and a 1-μg aliquot of purified total RNA was subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis using a SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). The resulting cDNAs were used as a template for PCR amplification to generate products corresponding to the mRNAs encoding various gene products. Each PCR reaction mixture contained cDNA, dNTP mix (Takara Biomedical, Shiga, Japan), 10× PCR buffer (Takara Biomedical), and Pyrobest PRKACG (Takara Biomedical). The cDNAs were amplified under the following cycling conditions: For GADPH, the cDNA was amplified with 30 cycles of denaturation at 94°C for 0.5 min, annealing at 60°C for 0.5 min, and extension at 72°C for 0.5 min; and for MMP-1, MMP-2, MMP-9, MMP-14, integrin α1, integrin α2, integrin α3, integrin α4, integrin α5, and integrin α6, the cDNA was amplified with 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 2 min were carried out. All PCR amplifications were performed using a DNA thermal cycler (Takara PCR thermal cycler MP; Takara Biomedical).