FIGURE 3.

The His pulldown assay and analytical ultracentrifugation analysis both confirm the Arn·H-NS interaction. A, C-terminal His₆-tagged Arn serves as bait, and tag-free H-NS serves as prey in this His pulldown assay. The His pulldown lanes, lane 1–3, are His pulldown eluates that represent the status of H-NS, Arn-His, and H-NS/Arn-His binding to nickel beads or protein complex pulled down, respectively. The input lanes, lane 4 and 5, are purified Arn-His and H-NS protein that are used in the assay respectively. B, under the buffer conditions of 20 mm Tris at pH 7.5 and 50 mm NaCl, the protein distribution of AUC analysis was as follows: the H-NS DNA binding domain (we used the protein His-H-NS91; the theoretical molecular mass is 8 kDa) only exhibits an 8-kDa peak as a monomer; the Arn protein (we used Arn-His; the theoretical molecular mass is 12 kDa) exhibits a single 21.6 kDa peak as a dimer; the His-H-NS91 and Arn-His mixture shows an ∼8-kDa peak that overlaps with the peak of the His-H-NS91 monomer and an ∼26.2 kDa peak that is significantly larger than the dimer peak of Arn-His.