Rabbit skeletal muscle globular actin (G-actin) was resuspended in GAB buffer (5 mM Tris-HCl pH 8.0, 0.2 mM CaCl2, 0.5 mM DTT, 0.2 mM ATP) as prescribed (Cytoskeleton). G-actin concentration was determined by absorbance at 290 nm (with the extinction co-efficiency of 26,600). We polymerized 4 μM G-actin by addition of salts (50 mM KCl, 2 mM MgCl2, 1 mM ATP) for half an hour at room temperature. Actin bundling assay was performed by mixing F-actin with tested proteins at indicated concentration and stayed in room temperature for 1 hr. Before observation by fluorescence microscopy, the mixture was labeled by AlexaFluor 488-Phalloidin (Thermo Fisher Scientific) and transferred to a coverslip. Images were taken by M2 upright microscope (Zeiss).

The quantification of the length and number of actin bundles was performed followed the previous description (Breitsprecher et al., 2008). Briefly, we polymerized 1 μM G-actin by the addition of salts (50 mM KCl, 2 mM MgCl2, 1 mM ATP) for 10 min at room temperature and then mixed with buffer, 5 μM IPP protein, or the IPP/Rsu1 mixture. After labeling by AlexaFluor 488-Phalloidin, the samples were diluted 20 times with the GAB buffer plus salts (50 mM KCl, 2 mM MgCl2, 1 mM ATP), and 5 μl samples were loaded to the coverslip for observation. Twenty images were taken for each sample by M2 upright microscope (Zeiss). The number and length of actin bundles were measured in ImageJ using Multi-point and Freehand Line tools.

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