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. 2018 Oct 30;293(51):19785–19796. doi: 10.1074/jbc.RA118.003784

Figure 2.

Figure 2.

F-actin–binding properties of ExoY variants. A, SDS-PAGE analysis of supernatant (sup) and pellet (pel) fractions after ultracentrifugation of 2 μg (3 μm) of ExoY variants in the absence or presence of actin. The upper band corresponds to ExoY, and the lower band corresponds to actin. B, the fractions of ExoY variants that cosedimented with F-actin were quantified by densitometry using ImageJ software and plotted against actin concentrations. Error bars correspond to standard deviations of three independent experiments. C, pulldown experiment with F-actin and ExoY variants performed as follows: 12 μg of ExoY variants and/or 12 μg of F-actin were added to nickel–nitrilotriacetic acid–agarose beads and incubated for 1 h at 4 °C. The samples were washed three times with a buffer containing 20 mm imidazole and eluted by addition of hot Laemmli buffer. Corresponding amounts of bound (El) and unbound (flow through, FT) fractions were analyzed by SDS-PAGE, stained with Coomassie, or blotted and incubated with anti-actin antibodies (WB, anti-actin C4 Abcam). The upper band corresponds to ExoY, and the lower band corresponds to actin. The figure is composed of three parts of two gels corresponding to the same experiment. D, SDS-PAGE analysis of supernatant and pellet fractions after ultracentrifugation of 2 μg (3 μm) of ExoY variants in the absence or presence of actin in high ionic strength conditions. The upper band corresponds to ExoY, and the lower band corresponds to actin. In this set of experiments, ExoY proteins (3 μm) were incubated with actin (indicated concentrations) polymerized to a steady state in the presence of 150 mm Tris, pH 9.0, 360 mm NaCl, 20 mm MgCl2, 1 mm DTT, and 2.5 mm ATP. These experimental conditions were chosen to maintain ExoY variants in a soluble form and prevent nonspecific aggregation/sedimentation. E, the fractions of ExoY variants that cosedimented with F-actin were quantified by densitometry using ImageJ software and plotted against actin concentrations. Error bars correspond to standard deviations of three independent experiments.