FIG. 2.

In vitro analysis of the interactions of VirB10. Interaction of VirB10 with VirB8, VirB9, and VirB10 was monitored by the GST-pulldown assay (2). Purified histidine-tagged VirB10 was incubated with GST or a GST fusion protein immobilized on glutathione-Sepharose. Binding of His-VirB10 was determined by analysis of bound proteins by SDS-polyacrylamide gel electrophoresis, followed by Western blot analysis using anti-VirB10 antibodies. Lanes 1 to 4, protein bound to immobilized GST, GST-VirB10, GST-VirB8, and GST-VirB9, respectively; lanes 5 to 8, the corresponding unbound proteins; lane 9, immobilized GST-VirB10 incubated with buffer only. The higher-molecular-weight band in lane 2 is the GST-VirB10 fusion protein. The His-VirB10 protein is indicated by the arrowhead.