ROCK1 preferentially localizes to actomyosin filament bundles, whereas ROCK2 also localizes to membrane protrusions. (A) Representative CHO.K1 cells expressing either ROCK1-GFP (top two rows) or ROCK2-GFP (bottom two rows) and costained for rhodamine phalloidin. Ratiometric images on the right were created by dividing the ROCK-GFP signal by rhodamine phalloidin; intensity was inverted so that areas of greatest actin association are brighter. f indicates actomyosin filament bundles; p indicates protrusions. Arrows indicate actin filament bundles at the rear of the cell, and arrowheads indicate actin filaments within protrusions. (B) Quantification of the mean intensity of the ratiometric image in protrusions versus actomyosin filament bundles at the rear of the cell. ROCK2-GFP similarly localizes to both actin filaments in protrusions and actomyosin filament bundles, resulting in a ratio approaching 1. n = 14 ROCK1-GFP– and 14 ROCK2-GFP–expressing cells. Error bars indicate SEM.