Assaying activities of small GTPases. Bone marrow–derived neutrophils were prepared from tamoxifen-induced (KO) and mock-induced (WT) Arap3^fl/flERT2Cre⁺ mice. Neutrophils were or were not plated onto polyRGD. Ten minutes later, cells were lysed using ice-cold lysis buffer. (A-B) Clarified lysates were used for pull-down assays to determine GTP-bound fractions of small GTPases: Rap1 pull-downs using GST-Ral GDS beads are shown in panel A and Arf6 pull-downs using GST-MT2 beads are shown in panel B. After incubation of baits with lysate, beads were washed, boiled in sample buffer, and proteins were subjected to SDS-PAGE and immunoblotted using anti-Rap1 (A) and anti-Arf6 (B) antibodies. Graphs show pooled data (means ± SEM) obtained from 3 independent experiments. Photographs of representative experiments are shown. Two different exposure lengths of the same blot are shown in the Arf6 pull-down panel because there was a large difference between unstimulated and stimulated samples (dotted line). For the same reason, the 2 conditions were quantified independently. (C) Clarified lysates were used for RhoA G-LISA assays. To allow comparison of 4 independent experiments, suspension readings were equalized (left graph). Statistical analyses were carried out with the raw data using 2-way ANOVA with Bonferroni post tests or paired t tests for pairwise comparisons. *P < .05.