Figure 3: Screening LCN2 mutant proteins for urinary excretion.

(a) Top panel: mouse urine was collected over 3 h after the inoculation of human LCN2 monomers and dimers of mutant proteins were excreted into the urine, whereas native human LCN2 was not excreted. The urine was analysed using anti-human LCN2 antibodies under non-reducing conditions to visualize both monomeric and dimeric species. Middle panel: all of the LCN2 species were immunoreactive with human specific LCN2 antibodies. Reducing conditions. Bottom panel: as a loading control, each mutant was also detected by Coomasie staining (100 ng per lane); reducing conditions. (b) Comparison of native, K3 and K3Cys mutants. Native and K3 formed dimers, but K3Cys produced only monomers in vitro (left panel) and in vivo (right panel). K3 and K3Cys were excreted to a much greater extent than native LCN2. Urine was collected for 3 h after i.p. innoculation (100 μg in 100 μl PBS, right panel). Non-reducing conditions. Anti-human LCN2 antibodies. (c) Urinary excretion of Alexa568-labelled native, K3 and K3Cys proteins (100 μg protein per mouse with equal fluorescent intensity). The image shows urine collected from 0–20 min, and from 20 to 180 min. (d) Urine was collected over 6 h and LCN2 was quantified by immunoblot. Note that the excretion of K3Cys exceeded native LCN2 by nearly 10-fold (72±19%; n=68 versus 4.9±3%; n=6; P=0.0001). Mean±s.d. Statistical analysis was performed by Student’s t-test.