NIH3T3 cells stably expressing fusions of a constitutively active small GTPase to the L106P destabilizing domain were split into three pools. The first population (−) was mock-treated and the second population (+) was treated with 1 mM Shld1 for 24 hr. The third population (+/−) was treated with 1 mM Shld1 for 24 hr, then washed with media and cultured in the absence of Shld1 for 24 hr (RhoA Q63L) or 48 hr (Cdc42 Q61L, Arl7 Q72L). Cells were serum-starved for 12 hr, fixed, stained with Alexa Fluor 488-conjugated phalloidin, and visualized using confocal microscopy.