RhoA activity assay

RhoA activity was measured by using a RhoA G-LISA absorbance-based biochemical assay kit (Cytoskeleton) according to the manufacturer's instructions. In brief, cells were lysed, aliquoted and snap frozen in liquid nitrogen. After rapid thawing, binding buffer was added to the cell lysate, which was subsequently incubated on a RhoA-GTP affinity plate coated with RhoA-GTP-binding protein in each well. The plate was placed on an orbital plate shaker at 400 rpm for 30 min at 4°C. After washes, primary anti-RhoA antibodies (1:250) and secondary HRP-linked antibodies (1:62.5) were sequentially added to the wells followed by an incubation on an orbital shaker at 400 rpm for 45 min at room temperature. Thereafter, the signal was developed with HRP-detection reagents. The absorbance was measured by means of a plate reader spectrophotometer Enspire (PerkinElmer). In the quantifications, the absorbance values of control cells were set to 1 in each experiment, and the differences between the control and the knockout, knockdown or transfection cells in corresponding experiments were calculated. The statistical differences between the two groups were assessed using the paired t-test. P<0.05 was considered significant.