Rac1/Cdc42 activity assay

Previously sorted GFP and mCherry positive cells were seeded, stimulated and recovered from matrigel. Recovery from matrigel utilized ice cold PBS and gentle mechanical separation by pipette. Cells were centrifuged at 1000xg for 1 min and washed. Cdc42 Activation Assay Biochem Kit (Cytoskeleton, Inc., Denver, CO, USA) was used to process cells. For each pulldown sample, 0.5 mg/mL total cell lysate was incubated with 10 ug PAK-PBD beads and the assay protocol was followed according to kit instructions. Samples were run on a 4-12% Bolt Bis-Tris Plus SDS-PAGE gel (Novex, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA), transferred to PVDF membrane and blotted for Cdc42, Rac1, XB130 and Tks5.