FIG. 3.

Cleavage and clearance of WT-PA and Ub-PA. (A) Concentrations of PA83 and PA63 detected in plasma collected at 2, 6, and 24 h after injection of PA (100 μg) measured by 14B7 and 1E9 ELISA (n = 16 mice per group). Western blot shows samples from two mice probed with anti-PA polyclonal (1:5,000). For Western blotting, plasma samples were diluted 50-fold (2 h) or 10-fold (6 h) prior to loading 5 μl/well. (B) Ub-PA83 and Ub-PA63 detected in plasma and serum collected at 2 h and 6 h after injection of Ub-PA (100 μg) by Western blots probed with anti-PA polyclonal (1:5,000). Samples were diluted 50-fold prior to loading 5 μl/well. (C) Comparison of PA83 plus PA63 measurements for WT-PA and Ub-PA in plasma at 2, 6, and 16 h after injection of 100 μg (i.v.) into BALB/cJ mice. The combined PA83 and PA63 measurements for samples from mice injected with WT-PA were assessed by 14B7 ELISA (n = 16 for 2 h and 6 h and n = 2 for 16 h). The combined PA83 and PA63 measurements for samples from mice injected with Ub-PA were assessed by 1G3 ELISA (n = 8 for 2 h and 6 h and n = 4 for 16 h) and are likely to be underestimates due to the low sensitivity of this ELISA compared to the 14B7 ELISA.