GST pull-down validation of novel interactions identified by yeast mating assay.

GST pull-down results for six pairs of interaction are shown as labeled. Purified GST-tagged Y. pestis T3SS proteins were immobilized onto the glutathione sepharose beads 4B and incubated overnight with the putative interacting proteins that were identified by yeast mating assay. After extensive washing, the beads were added with 2×SDS buffer and boiled, and the proteins were separated by SDS-PAGE electrophoresis. The separated proteins were transferred onto the PVDF membrane followed by immunoblotting using anti-GST or anti-His antibodies. The top panel shows the His-tagged proteins that were loaded in each pull-down assay for GST or GST-tagged Y. pestis protein. The middle and the lower panels show the pull-down proteins.