The mAbp1 interacting protein HPK1 was involved in induction of adhesion of murine PMNs under flow conditions in vitro. (A) Western blot of whole-cell lysates and co-immunoprecipitates of wild-type dHL-60 cells and dHL-60 cells expressing an mAbp1-GFP construct (dHL-60-mAbp1-GFP) upon specific mAbp1-GFP pulldown with GFP-nanotraps. Cells were either exposed to immobilized fibrinogen (250 µg/mL) and treated with Mn²⁺ (1 mM) for 15 minutes at 37°C or left in suspension without stimulation. mAbp1, HPK1, and actin were detected by immunoblotting by using specific antibodies. Western blot is representative for 3 independent experiments. (B,C) Confocal microscopy of dHL-60-mAbp1-GFP cells stained for HPK1 and actin. Cells were treated with Mn²⁺ (1 mM) and exposed to immobilized fibrinogen (250 µg/mL) (B upper panel), or were seeded on coverslips coated with ICAM-1 (12.5 µg/mL) and IL-8 (1 µg/mL) (B lower panel, and C) for 15 minutes at 37°C. After fixation and staining, confocal laser scanning microscopy was conducted. (B) Colocalization (yellow) of mAbp1-GFP (green) and HPK1 (red) at the lamellipodium (arrowheads) of polarized dHL-60 cells. (C) Colocalization (white) of HPK1 (red) and actin (cyan) at the lamellipodium (arrowheads) of polarized dHL-60 cells. Images are representative of at least 3 independent experiments; bar = 10 µm. (D) Induction of adhesion of isolated murine PMNs under flow conditions (1 dyne/cm²). Flow chambers were coated with P-selectin (10 µg/mL), ICAM-1 (12.5 µg/mL), and CXCL1 (5 µg/mL). PMNs were treated with a nonbinding control antibody (crtl immunoglobulin G [IgG]), or function blocking anti–Mac-1, or anti–LFA-1 antibodies (30 µg/mL) as indicated. Numbers indicate relative PMN adhesion after 10 minutes in percent of all interacting PMNs (100%). n = 4; mean ± standard deviation (SD); *P < .05; **P < .001; n.s. = not significant.