Universal hood 3 molecular imager

Bacteria cultures were adjusted to the same OD₆₀₀ and harvested by centrifugation. The bacterial pellets were resuspended in 1.5× SDS‐PAGE sample buffer and boiled for 5 min. Proteins were separated on SDS‐PAGE gels and transferred to nitrocellulose membranes (Amersham Protran). Membranes were blocked for 1–2 h at room temperature in Tris‐buffered saline with 0.1% (v/v) Tween 20 and 5% (w/v) milk powder (TBST‐milk), then incubated overnight at 4°C in TBST‐milk with the following primary antibodies: mouse anti‐FLAG M2, 1:5000 dilution (Sigma‐Aldrich); mouse anti‐DnaK, 1:10,000 dilution (Enzo); and mouse anti‐MBP, 1:15,000 dilution (Bio‐Rad). The next day, membranes were washed three times in TBST, followed by incubation with a secondary goat antimouse HRP, 1:10,000 dilution (Cell Signaling) in TBST‐milk at room temperature for 1 h. For detection, the membrane was incubated with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) for 5 min and imaged using a Bio‐Rad Universal Hood III Molecular Imager.

EMSAs were performed by mixing approximately 100 ng of each DNA fragment with increasing concentrations of purified MBP‐FoxR or MBP‐FoxR_G300S proteins in a binding buffer containing 10 mM Tris–HCl pH 8.0, 50 mM KCl, 1 mM DTT, 0.5 mM EDTA, 10 μg/ml of BSA, and 5% (v/v) glycerol. These reaction mixtures were incubated for 20 min at 26°C, then loaded on a 6% nondenaturing polyacrylamide gel in 0.5xTris‐borate‐EDTA (TBE) buffer, and electrophoresis was carried out at 80 V over 3 h. In order to pinpoint the binding site of FoxR, we performed EMSAs with 288 fmol of each DNA fragment in the reaction mixture. To study the effect of metal ions on DNA binding to MBP‐FoxR protein, 100 μm, or 10 μm of freshly prepared metal salts were added to the EMSA mix without EDTA and the reaction mixtures were incubated for 20 min at 26°C and then treated as above. To test the effect of siderophores on MBP‐FoxR‐DNA binding, either 10 or 100 μM of DFOB or DFOE (dissolved in DMSO) was added to the EMSA reaction mix. An equal volume of DMSO was included for the vehicle control. For all EMSAs, DNA bands were stained with ethidium bromide and visualized with a Bio‐Rad Universal Hood III Molecular Imager.