Figure 3

Cucurbitacin compounds react with Cofilin1 and inhibit activity. A) Ultracentrifugation was used to separate actin into soluble (S) monomeric actin and pellet (P) F-actin high speed fractions. Coomassie staining revealed that the addition of increasing Cofilin1 concentrations shifted actin from P to S fractions, indicating increased actin severing. The 5 μM Cofilin1 concentration was used for subsequent experiments. B) Cofilin1 at 5 μM was incubated with cucurbitacin E (CuE) at the indicated molar ratios. Protein mobility was slowed at 1:50 and 1:100 ratios. C) Incubation with cucurbitacin D (CuD), E or I (CuI) slowed Cofilin1 electrophoretic mobility, while co-incubation with 5 mM DTT blocked this effect on Cofilin1 mobility. D) Mobility shift of Cofilin1 could not be blocked if 5 mM DTT was added after incubation with cucurbitacin E. E) Actin partitioning into soluble (S) and pellet (P) fractions after ultracentrifugation was used to determine how actin-severing by Cofilin1 was affected by Cucurbitacin E. F) Quantification of the proportions of soluble (S) versus pelleted (P) actin fractions following ultracentrifugation for the indicated conditions. Cofilin1 increased the soluble monomeric actin fraction relative to control. Cucurbitacin E significantly (p = 0.05) inhibited Cofilin1-mediated actin severing relative to control DMSO vehicle when the P fractions were compared by Student’s t-test (n = 3).