Cleavage by RavK inhibits actin polymerization.

A. Cleavage by RavK inhibited actin polymerization in actin sedimentation assay. 30-μg non-muscle actin (Cytoskeleton, 99% purity) was incubated with 5-μg wild-type RavKΔC50 or the catalytically dead mutant RavK_H95AΔC50 at 23°C for 2 h. The mixtures were precleared by centrifugation at 100,000g for 30 min and further used in actin sedimentation assays. Actin polymerization was allowed to proceed for 60 min, followed by ultracentrifugation at 100,000g for 40 min. The resulting supernatants and pellets were analyzed by SDS-PAGE followed by Coomassie brilliant blue staining. Groups: i, actin treated with RavKΔC50; ii, actin treated with RavK_H95AΔC50; iii, actin without treatment. Lanes: I, input; P, pellet; S, supernatant. B. Quantification of the percentage of polymerized actin versus total actin. The band intensity was quantified with ImageJ. All results are obtained from three independent experiments. Error bars represent standard error of the mean (SEM). **, p<0.01. C. Cleavage by RavK inhibited actin polymerization in a kinetic assembly assay. Non-muscle actin treated by RavKΔC50, RavK_H95AΔC50 or a control reaction with RavK was precleared by centrifugation at 100,000g for 30 min. 2.7-μM differently-treated actin and 0.3 μM pyrene-labeled actin were mixed and the fluorescence intensity of pyrene (arbitrary units, a.u.) was plotted versus time after the addition of a polymerization buffer to initiate the polymerization. D. Actin used in pyrene-labeled actin polymerization assay. Actin samples receiving indicated treatment were resolved by SDS-PAGE and detected by Coomassie brilliant blue staining.