Enrichment of Active Cdc42 from Cells

Cells were cultured for 96 h in 3D collagen matrices, and GTP-loaded Cdc42 from cell extracts was enriched by pull down with immobilized PBD domains from Pak1 (Kutys and Yamada, 2014 (link)). Briefly, endothelial cells were lysed by sonication in ice-cold lysis buffer containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.6, 1% Triton X-100, 150 mM NaCl, 5 mM ​MgCl₂, 1 mM dithiothreitol (DTT), Sigma-Aldrich protease and phosphatase inhibitor cocktail, and 5% glycerol. After high-speed centrifugation for 10 min at 4°C, supernatants were collected and protein concentration determined by the Bradford method. Next 500 μg of protein was incubated in the presence of 40 μg of PBD_Pak1–glutathione S-transferase protein beads (Cytoskeleton) for 1 h at 4°C. Beads were washed twice with 500 μl of ice-cold wash buffer (25 mM Tris-HCl, pH 7.5, 30 mM MgCl₂, 40 mM NaCl, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 μg/ml aprotinin, and 1 μg/ml leupeptin) and boiled in sample buffer (40 μl of beads in wash buffer plus 10 μl of 5× sample buffer) for 5 min. Samples were resolved by SDS–PAGE, followed by Western blotting. Membranes were probed with an anti-Cdc42 antibody (Abcam).