Active cdc42 pull down and detection kit

The activity of Cdc42 was measured using an active Cdc42 pull-down and detection kit performed according to the manufacture's protocol (16119, Thermo Fisher Scientific). Briefly, 10-15×10⁶ cells were untreated or treated with 1 µM cytarabine for 1 h, followed by cellular lysis using the lyse/wash/binding buffer provided in the kit after addition of 1x EDTA free inhibitor cocktail (Thermo Scientific). The lysates were centrifuged for 15 min at 16 000 x g at 4°C. Protein contents were measured by the Bradford protein assay. 800 µg protein of each condition was used for the pull-down with glutathione resin and agarose beads together with 20 µg GST-PAK1-PBD. The GTPγS and GDP controls were performed according to manufature's protocol. About 30 µl of pull-down samples were applied onto an SDS-gel and Cdc42 was detected by immunoblotting using the anti-Cdc42 antibody provided in the kit.

Activation of Cdc42 was measured in mock or HSV-1-infected HeLa monolayers at 15, 30, and 60 min pvi using the Active Cdc42 Pull-Down and Detection Kit (Thermo Scientific) according to the manufacturer's instructions.

PC-3/N and PC-3/C cells were grown in 10-cm cell culture dishes in serum free medium. Total protein was harvested using RIPA lysis buffer containing protease inhibitor cocktail. The small Rho GTPase pull down assays were performed using active Rho detection kit (Cell Signaling Technologies), active Rac1 pull-down and detection kit and active Cdc42 pull-down and detection kit (Thermo Fisher Scientific) according to manufacturer instructions.