Serpin B2, also known as PAI-2, is an approximately 60 kDa serine protease inhibitor. It is primarily secreted by macrophages and monocytes and can form disulfide-linked multimers. Serpin B2 inhibits both the urokinase-type and tissue-type plasminogen activators (uPA and tPA). Serpin B2 also promotes the clearance of uPA by enhancing its binding and uptake by LRP. It limits fibril formation by Huntington protein (HTT) and beta-Amyloid peptides. It promotes Th2 biased immune responses and is important for intestinal CCL2 production, monocyte recruitment, and nematode clearance. A non-glycosylated form of Serpin B2 is retained intracellularly where it interferes with TNF-a induced apoptosis by protecting the Retinoblastoma protein (RB1) from calpain digestion. It also inhibits proteasome activity in activated endothelial cells. The activity of recombinant mouse PAI-2 was measured by its ability to inhibit uPA cleavage of a peptide substrate, N-carbobenzyloxy-Gly-Gly-Arg-7-amido-4-methylcoumarin (Z-GGR-AMC) in the assay buffer 50 mM Tris, 0.01% Tween 20, pH 8.5. The 50 ul different concentrations of rmPAI-2 (MW: 47.9 KD) was incubated with 50ul 2ug/ml rhuPA (EPA140Mu61) at room temperature for 15 minutes. Loading 50 µL of the incubated mixtures into empty wells of a plate, and start the reaction by adding 50 µL of 200 µM substrate (Z-GGR-AMC). Include a substrate blank containing 50 µL of assay buffer and 50 µL of 200 µM substrate. Then read at excitiation and emission wavelengths of 380 nm and 460 nm, respectively, in kinetic mode for 5 minutes. The result was shown in Figure 1 and it was obvious that PAI2 significantly decreased uPA activity. The inhibition IC50 was <300 nM.