561979.fig.001
Figure 1: Recognition of TvLEGU-1 by the anti-TvLEGU-1r antibody in total protein extracts and protease-rich extracts of T. vaginalis. (A) Coomassie brilliant blue-stained purified recombinant TvLEGU-1r protein (CBB; lane 1). WB assays of TvLEGU-1r incubated with the anti-TvLEGU-1r antibody (lane 2) or with the PI serum (lane 3) used as a negative control, both at 1 : 40000 dilution. Arrowhead points to the recombinant protein band TvLEGU-1r (~46-kDa). kDa, molecular weight markers in kilodaltons (Bio-Rad). (B) Coomassie brilliant blue-stained protein patterns of trichomonad protease-rich (lane 1) or total protein extracts (lane 3); WB assays of duplicated gels transferred onto NC membranes containing trichomonad protease-rich (lane 2) or total protein extracts (lane 4) incubated with the anti-TvLEGU-1r antibody, or with the PI serum (lane 5) used as a negative control, both at 1 : 20000 dilution. Arrowheads show the position of the native TvLEGU-1 (~30- and ~20-kDa) proteins. Asterisk shows a light protein band of ~60-kDa also detected by the anti-TvLEGU-1r antibody. kDa, molecular weight markers in kilodaltons (Bio-Rad). (C) Silver-stained 2DE protease-rich extracts from parasites grown in regular medium (panel a). WB of duplicate gels transferred onto NC membranes incubated with the anti-TvLEGU-1r antibody (panel b) or with the PI (panel c) serum both at 1 : 5000 dilution. Numbers 1–3 show the position of the TvLEGU-1 protein spots. (D) Differential phosphorylation of TvLEGU-1. 2DE WB assays from duplicate protease-rich extracts from part (C) separated by SDS-PAGE were (a) silver-stained or transferred onto NC membranes and incubated with the (b) antiphosphoserine (α-PSer), (c) antiphosphothreonine (α-PThr), and (d) antiphosphotyrosine (α-PTyr) monoclonal antibodies. pI, direction of IEF using IPG Ready-strips (linear gradient of pH 4–7; Bio-Rad). Numbers show the position of the TvLEGU-1 spots. (E) Landscape representation of the densitometric analysis (PDQuest and Melanie software) of the protein spots corresponding to TvLEGU-1. Numbers 1–3 show the position of the TvLEGU-1 spots.