Rho pull-down assay

The RhoA activity was determined using a rhotekin Rho-binding domain (RBD) affinity precipitation assay, followed by immunoblotting using a RhoA antibody. Active RhoA was affinity purified with GST bead-bound Rhotekin, which only binds RhoA in its active GTP-bound form. Then, Western blot analysis was performed to detect both active levels and total levels using a RhoA-specific antibody. A NewEast Biosciences RhoA Activation Assay Kit (Malvern, PA, USA) was used to detect the levels. Briefly, ice-cold lysis buffer (25 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 1% Igepal CA-630, 10 mmol/L MgCl₂, 1 mmol/L EDTA, 10% glycerol, 1 Ag/mL aprotinin, 10 Ag/mL leupeptin, and 1 mmol/L Na₃VO₄) was added to brain homogenate (2:1 v:v), and incubated on ice for 30 mins. The lysates were centrifuged for 10 mins (12,000×g at 4°C). Lysates were incubated with agarose-conjugated rhotekin-RBD for 1 hr at 4°C and were then washed three times using lysis buffer. Agarose beads were boiled in SDS-PAGE sample buffer to release active Rho, and samples were resolved on a 12.5% polyacrylamide gel followed by immunoblotting using a RhoA antibody.