Rhoa rac1 cdc42 combo activation assay kit

HUVECs were seeded on a 6-well plate and grown in complete media. Eighty percent confluent cells were then starved for 3 h (0.5% FBS) and treated or not for 30 min with the anti-α5 antibodies (5 μg ml⁻¹) followed by the addition of 100 nM spike for an incubation of 30 min. Cells were washed twice with ice-cold PBS, and the RhoA–Rac1–Cdc42 activation was evaluated using the RhoA–Rac1–Cdc42 combo activation assay kit (Abcam) according to the manufacturer's instructions. The kit is based on the binding of the active form of RhoA to the Rho-binding domain of rhotekin and of active Rac1 or Cdc42 to the p21-binding domain of p21-activated protein kinase 1. Both, rhotekin and p21-activated protein kinase 11 binding domains are coupled to agarose beads, and the isolation and detection of the active forms of RhoA, Rac1, and Cdc42 were done under conventional bead precipitation and Western blot protocols using goat antimouse alkaline phosphatase secondary antibodies (Jackson ImmunoResearch; 1:5000 dilution) and a colorimetric detection kit (Bio-Rad). Total levels of RhoA, Rac1, and Cdc42 were measured by Western blot in the absence of the isolation step.