Impaired chemokine-induced Cdc42 activation in AQP3-deficient T cells. (a–c) Quantifications of Cdc42, Rac1, and RhoA activation. T cells were incubated with 250 ng/ml CXCL12 for 1 min, and GTP-bound active form of Cdc42 (a), Rac1 (b), and RhoA (c) were assessed using the G-LISA activation assay kit (SE; n = 4–5; *, P < 0.05; **, P < 0.01). (d) Immunoblot analyses to detect the phosphorylation of WASP and Arp2. T cells were incubated with 250 ng/ml CXCL12 for 3 min and were analyzed using antibodies against phospho-WASP (P-WASP), WASP, phospho-Arp2, Arp2, and β-actin. The blots shown are representative of three separate sets of experiments. (e) Phosphorylated Itk in response to CXCL12 (250 ng/ml, 1 min) was quantified with BD cytometric bead array (SE; n = 4–5; **, P < 0.01). (f and g) F-actin polymerization in response to 500 ng/ml CXCL12 in AQP3 knockdown (f) and/or V12-Cdc42–positive human primary T cells (g). One representative experiment of three experiments is shown. Experiments in a–c and e were performed in two other independent experiments.