On the other hand, when the probe was incubated with the anti-DNAB-II antibody without protein extract, neither shifted nor supershifted band was observed, ruling out nonspecific antibody-probe KU55933 clinical trial interactions. Furthermore, no supershifted band was revealed when unrelated antibodies were evaluated, again validating the specificity of the antibody used (see Additional file 1). These assays indicated that members of the DNAB-II family (IHF or HU) are involved in the protein-DNA complex that forms at the phtD promoter region. Finally, to provide additional confirmation that IHF or HU contributed to the gel mobility shift results, we performed

shift-western experiments, in which shifted bands were transferred to nitrocellulose membranes and incubated with anti-DNABII family protein antibodies. Incubation with antibodies yielded one band at a position identical to that of the shifted band (Figure 3C), supporting the presence of a DNAB-II family DNA-binding protein (IHF or HU) in the complex identified by gel mobility assays. IHF protein interacts with the phtD operon promoter region To determine the identity of the protein observed in gel shift assays, we analyzed crude protein extracts of E. coli single mutants having, deletions in the genes coding for EPZ-6438 order the alpha and beta subunits of IHF and HU proteins by gel mobility shift assays. The bacterial strains

were grown in LB at 37°C until the cells reached the early stationary phase, when IHF levels are CP-868596 manufacturer reported to increase and even small amounts of HU protein are observed [31]. Incubation of the P phtD probe with crude extracts from E. coli strains K12 wild type, hupA – , and hupB – , showed a retardation signal similar to that obtained with extracts of P. syringae pv. phaseolicola NPS3121, indicating that mutations in genes encoding HU Regorafenib manufacturer protein subunits have no effect on the presence of the putative phtD regulatory protein. However, when crude extracts of E. coli mutants ihfA – and ihfB – were assayed, no retarded signal was observed (Figure 4A). These results strongly suggest that the protein involved in the DNA-protein complex is IHF. To validate

these results, two types of additional experiments were performed: 1) mobility shift competition assays using the algD promoter region and 2) mobility shift assays with a complemented E. coli ihfA – strain. Figure 4 Gel shift assays using Escherichia coli mutant strains and purified IHF protein. Gel shift assays were performed as described in Methods. (A) Protein extracts of E. coli mutants for subunits of HU (hupA, hupB) and IHF proteins (ihfA and ihfB) were used in these assays. The arrow indicates the DNA-protein complex formed. (B) Gel shift assay using the purified IHF protein from E. coli (IHFr), which produces a retarded signal similar to that obtained with the extract of P. syringae pv. phaseolicola. The probe used in this assay corresponds to the 104 bp region.