Assay of Rac1 GTPase Activity

GTPase-linked immunosorbent assay was performed to determine Rac1 activation in vitro (Cytoskeleton, Denver, CO, USA). Podocytes were lysed in lysis buffer to prepare lysates by centrifugation at 10,000 g for 1 min at 4°C. Equalized amounts of protein (1 mg/mL) were loaded onto a Rac1 GTP affinity plate for 30 min. Following incubation, the plate was washed in wash buffer and then incubated with primary and secondary antibodies against Rac1. The activated Rac1 was determined after exposure to HRP detection reagents by measuring absorbance at 490 nm using a microplate spectrophotometer.