Surface PDI-αMβ2 interaction is enhanced on stimulated neutrophils. (A-B) Human neutrophils were incubated without or with fMLF. After SSB labeling, lysates were immunoprecipitated with anti-PDI antibodies and immunoblotted with indicated antibodies (total interaction). The blots were reprobed with peroxidase-conjugated avidin (surface interaction). (B) The band density was quantitated by densitometry (mean ± SD, n = 4-5). (C) Surface plasmon resonance assay was performed as described in “Materials and methods.” The extracellular domain of recombinant αMβ2 (25 μg/mL in 10 mM acetate buffer, pH 5.0) was immobilized on the surface of a CM5 chip. Recombinant PDI (0.03-21 μM) or purified FG (0.02-5 μM, data not shown) in running buffer (10 mM HEPES, pH 7.5, 150 mM NaCl, 0.005% P20 with 2 mM MnCl²) were infused over the reference and αMβ2-immobilized surfaces at a flow rate of 5 μL/min for 240 seconds, followed by a dissociation phase of 300 seconds. The representative graph is shown from triplicate. The dissociation constant, K_d, was calculated based on the K_on and K_off value. (D) WT and αMβ2 null neutrophils were stimulated with fMLF in the presence of 50 μg/mL His-tagged wtPDI or dmPDI. Binding of recombinant PDI was analyzed by flow cytometry as described in the panel G section of the Figure 2 legend. Data represent mean ± SD (n = 3-4). *P < .05 and **P < .01 vs unstimulated neutrophils, and #P < .05 vs PDI binding to stimulated WT neutrophils after the Student t test. (E) Using lysates of unstimulated and fMLF-stimulated neutrophils, fractions containing lipid rafts and nonrafts were collected and immunoblotted. Representative blots and densitometric analysis are shown (n = 3).