Figure 5.

In vitro interaction assays between His ₆ -Wzd (34 kDa) and GST-Wze (53 kDa) proteins. His₆-Wzd bound to Ni²⁺ charged resin was incubated with the cell lysate of GST-Wze in the presence of 10 mM ATP (lanes 1, 4, 7 and 10) or absence of ATP (lanes 2, 5, 8 and 11). Lanes 3, 6, 9 and 12 contain native His₆-Wzd alone on the Ni²⁺ resin. Interaction of GST-Wze with His₆-Wzd expressed by E. coli BL21(DE3): (A) Coomassie stained 12% SDS-PAGE gel and (B) Western blot probed with mouse monoclonal anti-phosphotyrosine antibody. Interaction of GST-Wze with resin-bound His₆-Wzd expressed by E. coli C41(DE3) and treated with YOP (Yersinia tyrosine phosphatase): (C) Coomassie stained 12% SDS-PAGE gel and (D) Western blot probed with mouse monoclonal anti-phosphotyrosine antibody.