Fig. 3. Slac2-a bound MC myosin-Va with higher affinity than it bound brain myosin-Va. (A) Recombinant MC myosin-Va-tail (left panel) and brain myosin-Va-tail (right panel) were systematically diluted as indicated, and diluted samples were incubated with beads coupled with T7-Slac2-a protein (0.25 µg). Proteins bound to the beads were analyzed by 10% SDS-PAGE and stained with Coomassie Brilliant Blue R-250 (top panels). The bottom panels indicate the input FLAG-myosin-Va-tail (1/50 volume of the reaction mixture) visualized with HRP-conjugated anti-FLAG tag antibody (1/10,000 dilution). The positions of the FLAG-myosin-Va-tail and T7-Slac2-a proteins are indicated by the closed arrowhead and open arrowhead, respectively. (B) Bands of myosin-Va-tail on gels were captured and quantified as described in Materials and Methods. The EC₅₀ value of 0.15 for the Slac2-a·MC myosin-Va-tail interaction was calculated with GraphPad PRISM software (broken line). Bars indicate the standard error of three independent experiments. Open circles, Slac2-a·MC myosin-Va-tail interaction; closed circles, Slac2-a·brain myosin-Va-tail interaction; open squares, Slac2-a-GT·MC myosin-Va-tail interaction; and closed squares, Slac2-a-GT·brain myosin-Va-tail interaction. Judging from the intensity of the bands in A, one Slac2-a molecule binds approximately two molecules of myosin-Va-tail. The results shown are representative of three independent experiments.