Assay for the ratio of G-actin to F-actin

The ratio of G-actin to F-actin was analyzed using a kit (Cytoskeleton) according to manufacturer’s instructions. Briefly, after treatment, cells were lysed in a detergent-based lysis buffer that stabilizes and maintains the G– and F– forms of cellular actin. Cell lysates were transferred to an ultracentrifuge and spun at 150,000×g for 1 h to separate the globular (G)-actin (supernatant) and filamentous (F)-actin fractions (Beckman). Samples of supernatant and pellet were electrophoresed on SDS-polyacrylamide gel. All samples were diluted with appropriate loading buffer and boiled for 5 min. Actin was quantified by Western blot analysis.