ARHGAP25 moderates CXCL12 signaling and is functionally affected by phosphorylation. (A) ARHGAP25 deficiency strengthens HSPC response to a CXCL12 gradient. HSPCs from Arhgap25^−/− or control mice were placed in the top wells of a transwell apparatus with CXCL12 in the bottom well (left), both wells (center), or neither well (right). After 1 hour, transmembrane migration was assessed by flow cytometry as described in “Methods.” Arhgap25^−/− HSPCs showed markedly increased migration across a membrane in response to a CXCL12 gradient (left), but not in the absence of a gradient (center, right). Results shown are representative of 3 independent experiments. (B) Arhgap25^−/− HSPCs were stained with antibodies to CXCL12 receptors to confirm that the augmented response to CXCL12 in Arhgap25^−/− HSPCs was not due to increased cell surface expression of CXCR4 or ROBO4. Shown are representative overlay histograms of CXCR4 (left) or ROBO4 (right) expression on Arhgap25^−/− LSKs (black traces) as compared with control LSKs (gray traces). MFIs and SDs are shown, as well as P value. N = 4 for each condition in this experiment, which was performed in triplicate. (C) Phosphorylation of GST-fused full-length (GST-WT) ARHGAP25 and its truncated fragments. Phosphorylation was performed using radiolabeled ATP and neutrophil cytosol as a kinase source, as described in “Methods.” Marked phosphorylation was observed in the full-length protein as well as in the PH and ID regions, whereas phosphorylation of GAP and CC domains was undetectable. (D) Mutation of S363 affects the ability of phosphorylated ARHGAP25 to inactivate Rac. GST-WT ARHGAP25 and GST-mutant (S363A) ARHGAP25 protein were phosphorylated with neutrophil cytosol and nonradiolabeled ATP. GTPase activation effect was measured 5 minutes after co-incubation with GST-Rac by nitrocellulose filter binding assay, as described in “Methods.” ctrl, control; MFI, mean fluorescence intensity.