G lisa kit

BMMSCs were cultured for 4 h in serum-free medium. Then, the cells were lysed in lysis buffer, and the activities of three Rho family proteins, RhoA-, Rac-1-, and Cdc42-GTP, were measured using G-LISA kits (Cytoskeleton) following the manufacturer's instructions.

The G protein activity was determined by G-LISA assay, which has been described in the literature [22 (link)]. The activities of RHOA-, RAC1-, and CDC42-GTP were detected using G-LISA kits (Cytoskeleton, USA) according to the manufacturer's instructions. Plates with HepG2 cells cultured with serum-free medium for 6 h were placed on ice. The cells were washed with precooled PBS three times and allowed to stand for 1 min, after which 100 μl of protein lysis buffer (M-PER mammalian protein extraction reagent, Thermo Fisher Scientific) (containing 1 mM of protease inhibitors, Thermo Fisher Scientific) was added to each well. Cells were collected and placed in 1.5 ml Eppendorf tubes for lysis on ice for 10 min. Next, 10 μl of lysates was added into the wells of a 96-well plate, to which 290 μl of reagents was added for protein analysis. The OD value was detected at 600 nm 1 min later, and the concentration value was calculated by subtracting the value in the blank control group.