Pin1 is expressed in human neutrophils and is activated by TNF-α and fMLF. (A) Recombinant Pin1 (0.5 μg) and a resting human neutrophil (PMN) lysate (2 × 10⁶ cells) were analyzed by SDS-PAGE and Western blot with an anti-Pin1 antibody. (B) Resting human neutrophils were lysed by nitrogen cavitation, and fractions were isolated on a Percoll gradient. Proteins were analyzed by SDS-PAGE and Western blot with an anti-Pin1 antibody. IB indicates immunoblot. (C) Recombinant Pin1 (0.25 nmol) was used to measure activity by recording the absorbance (at 390 nm) of free p-nitroaniline (pNA) cleaved by trypsin from Trp-Phe-Tyr-Ser(PO3H2)-Pro-Arg-pNA in the absence and presence of juglone (250nM). (D) Neutrophils were incubated in the absence and presence of juglone (250nM for 30 minutes) and then treated with TNF-α (20 ng/mL), fMLF (10⁻⁷M), or TNF-α, followed by fMLF (TNF-α + fMLF), before lysis. Pin1 activity was determined by measuring the absorbance of free pNA cleaved from Trp-Phe-Tyr-Ser(PO3H2)-Pro-Arg-pNA. Data are mean plus or minus SEM of 6 experiments. *P < .01 compared with resting neutrophils. §P < .01 TNF-α + fMLF compared with fMLF. #P < .001 with versus without juglone.