Nsc23766

Pak-PBD (p21-binding domain of p21 activated protein kinase 1) inserted into pGEX-2T (GE Healthcare, Little Chalfont, UK) was transformed into BL21. GST-PBD fusion protein expression was induced by 0.1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 4 h. Bacterial pellets were collected, lysed, and cleared by centrifugation, and the bacterial cell lysate obtained was incubated with glutathione beads (GE Healthcare) to affinity purify the GST-PBD fusion protein. Two micrograms of immobilized recombinant protein was incubated with either 25 μg or 100 μg C6 cell lysates for 1 h at 4°C to detect Rac1 and Cdc42, respectively. Beads were then washed with RIPA buffer, lysates were eluted in SDS sample buffer by heating at 65°C for 15 min in SDS sample buffer, and Rac1 or cdc42 bound to GST-PBD was detected by immunoblotting. The intensity of the signal was quantified by densitometric analysis. Relative GTPase activities were normalized to the value of the control cells. For some experiments, cells were treated with 100 μM of the Rac1 inhibitor NSC-23766 (Santa Cruz Biotechnology) for 24 h. For HGF activation assay, cells were serum starved overnight; this was followed by addition of 50 ng/ml HGF for 5 min.