PDI regulates sulfhydryl exposure of αMβ2 integrin during neutrophil activation. (A-C) Human neutrophils pretreated without or with an anti-PDI antibody were stimulated with fMLF, followed by labeling with SSB or MPB. Lysates were pulled down with avidin agarose beads and immunoblotted. (D-F) WT and PDI KO neutrophils were stimulated with fMLF and used in the same pulldown assay. (B,E) The free thiol contents of surface αM, β2, and PDI (band density of MPB labeling) were normalized to the surface expression of each protein (band density of SSB labeling). Quantitative graphs are shown as mean ± SD (5-6 independent experiments for human neutrophils and 3 independent experiments for mouse neutrophils [5 WT and 5 KO mice per experiment in each group]). (C,F) The fold change of free thiol levels during cell activation was obtained by dividing the normalized value in stimulated cells by that in unstimulated cells in each group (mean ± SD). The number greater or lower than 1 implicates that sulfhydryl exposure per exposed surface protein increases or decreases following fMLF stimulation. *P < .05 vs unstimulated and IgG- or vehicle-treated control (MPB/SSB = 1).