Fig. 8. Binding of LimC and LimD to F-actin. Co-sedimentation of (A)  GST–LimC, GST–LimD and (B) GST–LimC deletion constructs GST–CLIM and GST–NLIM-P with F-actin. G-actin (5 µM) from D.discoideum was incubated with the GST fusion proteins in polymerization buffer containing 2 mM MgCl₂, 100 mM KCl and 1 mM EGTA. Pellets (P) and supernatants (S) were separated by high-speed centrifugation, and the proteins in these fractions were analyzed by SDS–PAGE (12% acrylamide) and staining with Coomassie Blue. Controls for the fusion proteins alone were performed without the addition of G-actin in the reaction mixture. The presence (+) or absence (–) of G-actin in the reaction mixture is indicated on the top of the lanes. Arrows marked with C and D indicate GST–LimC and GST–LimD, respectively. Varying amounts of polymerized (P) and unpolymerized actin (S) in GST–LimC and GST–LimD panels might be due to different actin preparations.