Figure 2

Identification of pro-u-PA-binding proteins in FhNEJ-Teg. A, B FhNEJ-Teg proteins were resolved by 2D electrophoresis in duplicate so that proteins in one of the gels were visualized by silver stain (A), and the others were transferred onto a nitrocellulose membrane to detect pro-u-PA binding by standard ligand blot procedures (B). Protein spots are circled and numbered. C Abundance distribution of the most recurrently identified proteins in the pool of potential pro-u-PA binding proteins (circled spots). D, E Gene ontology analysis of the potential pro-u-PA-binding proteins identified by 2D-MS in the biological process (D) and cellular component (E) categories plotted according to node score. The values in parentheses indicate the node score/percent of sequences annotated in each category. F Binding of the recombinant proteins FhCB3 and FhCL3, FhGST and FhENO to pro-u-PA was detected via ELISA by coating wells with the recombinant proteins (1 µg) followed by incubation with increasing amounts of pro-u-PA. The data points indicate the means of three technical replicates ± SD, and the asterisks indicate significant differences between all the recombinant proteins and the negative control (1% BSA), with identical p values for each comparison (**p ≤ 0.01; **** p ≤ 0.0001; one-way ANOVA), except in the wells incubated with 0.0625 µg of pro-u-PA. In this condition, the difference between FhCB3 and 1% BSA was not significant. Note that some data points overlap: those representing rFhCB3 and 1% BSA (0.0625 µg of pro-u-PA), those representing rFhCL3 and rFhGST (0.0625 µg of pro-u-PA), and those representing rFhGST and rFhENO proteins (1 µg of pro-u-PA).