The PH domain of kindlin-3 is crucial for integrin activation and neutrophil adhesion. (A) K3KO HL-60 cells were reconstituted with WT kindlin-3 (K3KO_EGFP-K3) or K3 with PH domain truncation (K3KO_EGFP-K3delPH). The cells were sorted to match similar expression levels of EGFP. Flow cytometry with mAb24 and KIM127 antibodies was used to evaluate the IL-8–induced high-affinity integrin activation (B) and extension (C). The cell arrest assay (D) was performed on a substrate of P-selectin/ICAM-1 at a wall shear stress of 6 dyn/cm². (E) Epifluorescence (Epifluor.) or TIRF image of EGFP-K3 and EGFP-K3dPH cells after stimulation with IL-8. (F) Quantification of the MFI of EGFP-K3 and EGFP-K3dPH recruited to the membrane. Means ± SEM of 9 EGFP-K3– and 5 EGFP-K3dPH–expressing cells. (G) qDF imaging of HL-60 cells expressing kindlin-3 PH domain-EGFP (green, right), kindlin-3-TagRFP (not shown) and labeled with membrane dye CMDR (magenta, left). Bar represents 5 μm. (H) The EGFP intensity was normalized to the membrane intensity, as in Figure 2. **P < .01; ****P < .0001.