Rac-GTP pulldown assay

A Rac1 Pull-Down Activation Assay Biochem Kit was used to isolate active Rac from whole-cell lysate as described previously (Graziano et al., 2017). Briefly, dHL-60 cells were serum starved with RPMI medium lacking FBS for 1 h in the incubator at a density of 2×10⁶ cells ml⁻¹. After starvation, cells were pelleted and suspended in mHBSS, and plated on a fibrinogen-coated 100 mm tissue culture dish to attach for 30 min. fMLP was then added to the cells at a final concentration of 100 nM, then cells were lysed with ice-cold lysis buffer at indicated time points and collected by scraping. 10 µg PAK–GST beads were mixed with each sample and incubated at 4°C for 1 h. Protein beads were washed and processed for western blotting.