TAGLN2 directly binds to F-actin and weakly cross-links actin in bundles. (A) Actin was polymerized in APB and then incubated for 30 min with 8 µM TAGLN2, 1 µM α-actinin, and 1 µM BSA. After high-speed centrifugation (100,000 g), pellets were separated from supernatants and analyzed by SDS-PAGE. S, supernatant; P, pellet. (B) To generate standard curves, different amounts of actin (4 µM) and TAGLN2 (0.4–5.6 µM) were loaded on an SDS-PAGE gel, and the intensities of the bands were measured by a densitometer. The actin binding affinity was measured as described in the Materials and methods. (C) For actin bundling assay, various doses of TAGLN2 (8–24 µM) were incubated with F-actin (4 µM). After low-speed centrifugation (10,000 g), pellets and supernatants of the samples were analyzed. All data shown are representative of three independent experiments. M, molecular mass. (D) The reactants above C were examined via EM. Images shown are representative of three independent experiments. Bar, 100 nm.