FIG. 5.
Competition between RsbV and σB for binding to RsbW as analyzed by using a nondenaturing 10% PAGE gel stained with Coomassie blue. (A) A mixture of RsbW and RsbV was incubated for a few minutes at room temperature to allow the formation of an RsbW2-RsbV2 complex before being challenged by increasing concentrations of σB. Lane 1, 5 μM RsbW2-RsbV2 complex (the higher band corresponds to the [RsbW2-RsbV2]2 complex); lane 2, RsbW2-σB complex; lanes 3 to 7, 5 μM RsbW2-RsbV2 complex challenged with 5, 10, 20, 30, and 50 μM σB, respectively. (B) Reciprocal experiment. A purified RsbW2-σB1 complex was challenged with increasing concentrations of RsbV. Lane 1, RsbW2-σB1; lanes 2 to 5, 13.7 μM purified RsbW2-σB1 complex challenged with 4, 8, 16, and 32 μM RsbV, respectively.