Protein interaction assays

CHO-K1 cells were transfected with pEGFP-talin1_1–433, -talin1_1–446, -talin1_1–449, or their mutants. At 28 h post transfection, the cells were harvested in lysis buffer A (50 mM Tris-HCl pH 7.4, 1% NP-40, 150 mM NaCl, 1 mM EDTA and a protease inhibitor cocktail). Cell lysates were cleared by centrifugation and incubated with glutathione–Sepharose beads loaded with GST or GST–β-integrin tails at 4°C for 2 h. The beads were washed with the lysis buffer four times and resuspended in SDS sample buffer. Samples were analyzed using SDS-PAGE and transferred to nitrocellulose membrane for the detection of interacting proteins. The binding of purified His-tagged proteins to GST–β-integrin tails was performed in lysis buffer A containing 3 mg/ml BSA (Fig. 1D) or 0.5 mg/ml gelatin (Fig. 1I).