Cortactin is required for ICAM-1–triggered activation of RhoG. (A) Primary MLECs isolated from Cttn^WT/WT (WT) and Cttn^del/del (KO) mice were incubated for 30 min with beads loaded with either control antibody (IgG) or the mAb YNI against ICAM-1. Active RhoG was pulled down from cell lysates with ELMO-GST and detected in immunoblots (top). Total RhoG levels subjected to pull-downs were controlled by blotting aliquots of the cell lysates (bottom). Quantification of signal intensity of the pulled down material is depicted below. (B) MLECs from Cttn^WT/WT (WT) and Cttn^del/del (KO) mice transfected with WT RhoG or CA-RhoG were incubated for 20 min with mouse PMNs followed by fixation and immunofluorescence microscopy for ICAM-1. 109 PMNs (on WT cells with WT RhoG), 102 PMNs (on WT cells with CA-RhoG), 81 PMNs (on KO cells with WT RhoG), and 92 PMNs (on KO cells with CA-RhoG) were analyzed for the presence of surrounding ICAM-1 rings. (C) Transmigration of mouse PMNs through a TNF-activated monolayer of MLECs from Cttn^WT/WT (WT) and Cttn^del/del (KO) mice transfected with WT RhoG or CA-RhoG. The amount of transmigrated neutrophils is displayed as the percentage of total applied cells. Data are presented as mean ± SD. ***, P < 0.005. (A–C) Experiments were independently performed three times.