Figure 8.

Nbs are stable in plasma for at least 72 h. Nbs were incubated in heparinized mouse plasma at 37 °C until a maximum of 168 h. A, Western blot against HA tag was performed to detect Nb 39 and Nb 53 full-length and cleavage products. B, quantifications of full-length Nb band were normalized to the full-length band at 0h for each Nb. The means of 3–5 independent experiments ± S.D. are represented. Time factor was analyzed by two-way ANOVA followed by Sidak's multiple comparisons post hoc test. *, p < 0.05; ***, p < 0.001, as compared with the 0-h time point. Differences between the two Nbs were determined by two-way ANOVA but are not identified (n.s.). C, Nb incubation in PBS did not give rise to degradation products as detected by Western blot. D, incubation of Nbs in plasma at 37 °C until a maximum of 72 h did not affect the capability of those in binding EphA4 LBD. All values represent the percentage of Nb-EphA4 LBD binding respect to the 0-h time point. This assay was done in triplicate, and data are represented as the mean ± S.D.