Rho antibody

Rho activation was measured using Active Rho detection kit (Cell Signaling Technology) following the manufacturer's instructions. Briefly, HMEC‐1 cells were seeded in 75 cm² flask to grow to a 70%‐80% confluence and treated with 1 μM IMB5046 for 30 minutes, then exposed to 10 μM PF‐228 or not for 10 minutes. Subsequently, cells were lysed and incubated with GST‐Rhotekin‐RBD and glutathione resin to selectively isolate and pull down the active form of Rho. The active form was detected by immunoblotting using a Rho antibody (8789, Cell Signaling Technology). Densitometric values of immunoblots were obtained using ImageJ. The values were corrected by the density of total Rho and normalized to the control expressed as 1. The experiment was repeated three times.

Proteins were extracted from HBMECs or mouse brain tissues using Pro-PREPTM Protein Extraction Solution (Boca Scientific, Westwood, MA, USA). Lysates (20 µg per sample) with equal volumes of Novex^TM SDS sample buffer (Thermo Fisher, Waltham, MA, USA) and 2-ME were heated at 95 °C for 5 min and then loaded onto 4–20% Tris–glycine gels. After electrophoresis and transferring to polyvinylidene difluoride membranes (Thermo Fisher, Waltham, MA, USA), the membranes were blocked in Brock Ace (Bio-Rad, Hercules, CA, USA) for 15 min at room temperature. Membranes were then incubated overnight at 4 °C with an anti-AKAP12 antibody (1:5000, obtained from the Gelman Lab at Roswell Park Cancer Institute), phospho-MCL antibody (1:1000, Cell Signaling), Rho antibody (1:1000, Cell Signaling) or anti-β-actin antibody (1:5000, Sigma Aldrich, St. Louis, MO, USA) followed by incubation with peroxidase-conjugated secondary antibodies and detection by Pierce ECL Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA).