Fig. 5.

MtrC binds to both MtrE E434K and MtrD D405K/D406K/K948E. The purified NT-MtrC GST-tagged protein was immobilized on GST column beads, to which was added purified His-tagged MtrE E434K (B) or MtrD D405K/D406/K948E (A) prey protein. The beads were washed with Tris buffer, containing 0.1% w/v βDDM, then boiled at 90°C for 5 min to elute the complex. Western blotting, using antibodies to the His-tag, was used to detect the MtrE and MtrD prey proteins. As negative controls, either the bait protein or the prey protein was omitted from the assay (the presence and absence of proteins is indicated by ‘+’ and ‘−’). As shown in each assay when either of these proteins was omitted from the assay, the corresponding prey protein could not be detected. These assays indicate that MtrC interacts with MtrE E434K and MtrD D405A/D406A/K948K.