Cells were washed with ice-cold PBS and lysed in RIPA buffer containing protease inhibitors for 10 min on ice. Cells lysates were cleared by centrifugation and protein levels determined (Bradford assay). Samples were boiled in laemmli buffer with ß-mercapto-ethanol for 5 min and run on Tris-glycine gels, transferred to PVDF membrane, and probed. Proteins were visualized with fluorescent secondaries (IRDye 680RD Goat anti-Mouse IgG, Li Cor, 926-68070) or chemiluminescene (HRP-linked anti-mouse IgG or anti-rabbit IgG, cell signaling, 7076S and 7974S).
Active Rac1 was measured as described previously (23 (link)). Briefly, cells were lysed (50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 0.1 M NaCl, 1% NP-40, and 10% glycerol with protease inhibitors) and incubated with Glutathione S-transferase (GST) coupled to the p21-binding domain of Pak (PBD) to precipitate Rac-GTP while rotating at 4°C. Beads were then washed 4 times in lysis buffer and resuspended in SDS sample buffer and heated (5 mins 95°C). Samples were then loaded onto SDS-PAGE and processed for Western blotting.
Protein levels were quantified using ImageJ software to measure density of target proteins bands relative to a control protein (tubulin). Results were graphed and presented as bar graphs.
Active Rac1 was measured as described previously (23 (link)). Briefly, cells were lysed (50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 0.1 M NaCl, 1% NP-40, and 10% glycerol with protease inhibitors) and incubated with Glutathione S-transferase (GST) coupled to the p21-binding domain of Pak (PBD) to precipitate Rac-GTP while rotating at 4°C. Beads were then washed 4 times in lysis buffer and resuspended in SDS sample buffer and heated (5 mins 95°C). Samples were then loaded onto SDS-PAGE and processed for Western blotting.
Protein levels were quantified using ImageJ software to measure density of target proteins bands relative to a control protein (tubulin). Results were graphed and presented as bar graphs.