Cdc42 antibody

The protein lysates (30–50 μg/sample assessed by BCA protein assay, Pierce Chemical Co., IL) were subjected to 10% SDS-Tris glycine gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were incubated with polyclonal GIT1 antibody (Cell Signaling, 1:1000), Rac1 antibody (Millipore 1:1000), Cdc42 antibody (Cell Signaling, 1:1000), RhoA antibody (Cell Signaling, 1:1000), MYO18A antibody (Santa Cruz. 1:1000), SNX27 antibody (Santa Cruz. 1:1000) and α-tubulin (Cell Signaling, 1:5000), at 4°c overnight. Peroxidase conjugated anti-rabbit antibody (1:5000) was used as a secondary antibody. The membranes were developed by Renaissance protein detection kit (DuPont NEN, MA, USA).

HUVECs were seeded at a density of 2 × 10⁵ in a 100 mm dish. The next day, cells were transfected with empty vector, DIAPH1 WT, or DIAPH1 double negative mutant for 24 hr and further starved overnight. Then, cells were treated with PBS or CCN1 (10 ng/mL) for 30 min. Protein lysate was collected by centrifugation at 16,000 × g at 4C for 15 min. Active Cdc42 was extracted from the lysate per the manufacturer’s protocol. Briefly, an equal amount of protein was used for GTPase assay (Active Cdc42 detection kit, 8819, Cell Signalling Technology). GST-PAK1-PBD fusion protein was used to bind the activated form of GTP-bound Cdc42. The level of activated Cdc42 was checked by WB with Cdc42 antibody (Cell Signalling Technology).