D. ZF21, 5-caccgcagtgtgacgccaagtttgacgaatcaaacttggcgtcacactgc-3; FAK1, 5-caccgcatcttccagttacaaattccgaagaatttgtaactggaagatgc-3, and FAK2, 5-caccgccaacccatctgatggaagacgaatcttccatcagatgggttggc-3. shRNA expressing lentiviral vectors were generated and used according to the manufacturer’s instructions (Invitrogen). For rescue experiments of ZF21 knockdown, an expression construct encoding a mutant ZF21 mRNA refractory to shRNA was generated by site-directed mutagenesis, and the mutant protein was expressed with a Myc tag at the C terminus using the pENTR vector (Invitrogen). Immunofluorescence Microscopy Cells were fixed in ?20 C methanol for 10 min and permeabilized using 0.1% Triton X-100 in PBS for 10 min. After the samples were blocked in PBS containing 5% goat serum and 3% bovine serum albumin, they were incubated with primary antibodies. All primary antibodies were visualized with either an Alexa 488- or 568-conjugated goat anti-mouse or anti-rabbit antibody (Invitrogen). Images of cells were captured with Leica ASMDW with CCD camera (Leica). Cell Fractionation Cells were homogenized in a buffer containing 250 mm sucrose and 25 mm Tris-HCl, pH 7.5. After centrifugation at 8,000 for 15 min, the supernatants were centrifuged further at 105,000 for 1 h. The pellet was used as the membrane fraction and the supernatant as the cytoplasmic fraction. Adhesion Assay For the cell adhesion assay, the surface of 12-well plates was coated with poly-l-lysine, fibronectin, collagen I, or vitronectin at a concentration of 1 1 g/ml, and nonspecific binding sites were blocked with 0.5 mg/ml bovine serum albumin. Cells suspended in serum-free DMEM were seeded onto the plates at 3 104 cells per well and incubated for 30 min. After removal of nonadherent cells, adherent cells were trypsinized and counted. The effect of blocking of integrin-1 on cell adhesion was analyzed as follows. 96-Well plates were coated with poly-l-lysine, collagen I, fibronectin, or vitronectin (1 g/ml), BI 224436 and nonspecific binding sites were blocked with 0.5 mg/ml bovine serum albumin. Cells suspended in serum-free DMEM were seeded onto the wells (2 104 cells per well) and incubated for 30 min in the presence or absence of 1 g/ml integrin-1 blocking antibody. After removal of nonadherent cells by washing, the remaining cells were stained with Hoechst 33342. Finally, fluorescence was quantified in a FLUOstar OPTIMA (BMG LABTECH). Cell Migration Assay Both sides of transwells with 8-m pore size filters (Corning Glass) were precoated with fibronectin (5 g/ml) for 1 h at 37 C. DMEM (400 l) containing 10% fetal bovine serum was added to the lower chamber, and 200 l of a cell suspension (5 104 cells) was placed in the upper chamber. The plates were incubated at 37 C in a 5% CO2 atmosphere for 6 h. Cells in the lower chamber were then stained with 0.5% crystal violet solution and counted using a light microscope at 200 magnification. Values are averages of three fields. Cell Surface Protein Pulldown Assay A pulldown assay of cell surface proteins was performed as described previously (33). Briefly, cells were washed three times with chilled PBS containing 1 mm MgCl2 and 0.1 mm CaCl2 and then incubated with sulfo-NHS-biotin BI 224436 (Pierce) (0.1 mg/ml) in the same buffer at 4 C for 30 min. The reaction was terminated by incubating the cells with 100 mm glycine in PBS. The cells were lysed in TNETS buffer (25 mm Tris-HCl, pH 7.5, 150 mm NaCl, 2 mm EDTA, 1% Triton X-100, 1% SDS), and the biotinylated proteins were nicein-125kDa precipitated BI 224436 with streptavidin-conjugated Sepharose beads (Amersham Biosciences). The samples were analyzed by Western blot using each antibody. TIRF Microscopy For TIRF microscopy, living cells were observed using an APON 60OTIRFM.