Characterization of interactions determinants of S endodomain binding to ezrin.

<p>A. Sequences of the wild type (wt) and mutated S endodomain used in the GST-pull down analysis. Twelve GST fusion proteins were produced with mutations in the S endodomain. Δ indicates truncations and C indicates cysteine to alanine mutations (in bold) of cysteine clusters (1 to 4). B. & C. Effect of truncations or cysteine to alanine mutations. Vero E6 lysate was incubated with Glutathione-Sepharose beads coupled or not with GST, GST-S_endo_wt (B. & C.), GST-S_{endo Δ8}, and GST-S_{endo Δ19}(B.), GST-S_{endo C1}, GST-S_{endo C2}, GST-S_{endo C3}, GST-S_{endo C4}, and GST-S_{endo C1-4} (C.) using 1 or 5 µg of GST fusion proteins. D. Effects of cysteine to alanine mutations and truncations or point mutations (K1227A and T1220A). Vero E6 lysate was incubated with Glutathione-Sepharose beads coupled with either GST, GST-S_{endo wt}, GST-S_endo_{Δ8 C1-4}, and GST-S_endo_{Δ19 C1-4}, GST-S_endo_ΔC, GST-S_{endo K1227A}, GST-S_endo_T1220A using 1 µg of each GST fusion protein. 5 µL of cell lysate was used as input control for B. & C. (8% of volume used in each pull down); 10 µL of cell lysate was used as input control for D. (5% of volume used in each pull down). IB: Immunoblot. Results shown are representative of two independent experiments.