Fig 6.
Effect of Spx mutants on RNAP interaction in vitro. The σA-depleted RNAP (SAd-RNAP) or RNAP holoenzyme (Holo-RNAP) was incubated with SpxΔCHA, Spx(R91A)ΔCHA, or Spx(R92A)ΔCHA in binding buffer (10 mM Tris-HCl, pH 8.0, 100 mM KCl, 5 mM MgCl2). By pulldown assay with anti-HA affinity chromatography, the interaction between Spx mutants and SAd-RNAP (A) or Holo-RNAP (C) was analyzed by SDS-PAGE. The intensity of each subunit of RNAP holoenzyme (B) or SAd-RNAP (D) on the gel was quantified and normalized, and results are presented as a ratio to the intensity of SpxΔCHA. Abbreviations: I, input; FT, flowthrough; E, eluate. The band labeled “milk” is protein from the blocking agent (dissolved powdered milk).