Binding of EhCaBP3 to actin.

<p>(<b>A</b>) Co-sedimentation assay of EhCaBP3 with F-actin. Polymerized F-actin (5 µM) was incubated with purified recombinant proteins EhCaBP3 (5 µM, lanes 7, 8), EhCaBP1 (5 µM, lanes 3, 4) and EhCaBP2 (5 µM, lanes 5, 6) as indicated. EhCaBP3 and EhCaBP1 were also incubated with actin (lanes 11, 12). EhCaBP3 alone was incubated without actin (lanes 9, 10). Actin alone without CaBP3 (lanes 1, 2). Ultra-centrifugation (100,000×g) was performed to separate supernatant (S) and pellet (P) fractions, followed by SDS-PAGE. (<b>B</b>) EhCaBP3 binding to G-actin. 5 µM Actin was coated overnight on 96well plate at 4°C, followed by blocking with BSA. EhCaBP3 was added in increasing concentrations as indicated in the presence (5 mM CaCl₂) or the absence of Ca²⁺ (2 mM EGTA) and EhCaBP1 (5 µM) was used as a control. This was followed by incubation with anti-EhCaBP3 or anti-EhCaBP1 antibodies. The amount of bound protein was detected using anti-rabbit IgG-HRPO. Absorbance was determined at 405 nm. The histogram shows relative mean intensity ± SD of three independent experiments. (<b>C</b>) Effect of EhCaBP3 on actin polymerization. Pyrene-labeled actin was polymerized without and with indicated concentrations of EhCaBP3 as described in “<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003055#s4" target="_blank">Materials and methods</a>”. The fluorescence of pyrene was observed at 407 nm. (<b>D</b>) F-actin bundling in presence of EhCaBP3. Polymerized F-actin (3 µM) was incubated with EhCaBP3 (5 µM) in presence of 5 mM CaCl₂ and 2 mM EGTA for 2 h. The samples were then centrifuged at (10,000×g) for 15 min. Under this condition, unbundled F-actin remains in supernatant and bundled F-actin sediments. Both supernatant (S) and pellet (P) were analyzed by SDS-PAGE.