Cdc42 g lisa activation assay kit

PC-3 cells were seeded and grown to 70% confluence before being transfected with FGD4 siRNA or control siRNA. Cells were harvested at 48 h after transfection and subjected to CDC42 activation assay using CDC42 G-LISA Activation Assay kit (Cytoskeleton) according to the manufacturer’s protocol. Briefly, cell lysates were prepared and equal amounts of protein were added to the tubes containing GTP-binding protein and incubated with shaking at 4 °C. Unbound proteins were washed off and antigen presenting buffer was added in tubes and incubated at room temperature. Primary antibodies were added and incubated at room temperature. Next, tubes were washed and secondary antibodies were added to them. Samples were incubated at room temperature, washed and HRP detection reagent mix followed by HRP stop buffer were added and absorbance of the developed color was detected in a spectrophotometer at 490 nm.