VapB and VapC interact <i>in vivo</i> and <i>in vitro</i>.

<p>(<b>A</b>) Pull-down assay. The soluble fraction of <i>E. coli</i> pAE<i>vapBC</i> extract was applied to a Ni⁺²-Sepharose column. Samples were analyzed by SDS-PAGE. Lane 1: initial sample; lane 2: washing; lanes 3–4: elution with 250 mM imidazole. It is important to observe that no VapC was released during the washing step, being co-purified with VapB-His, denoting the <i>in vivo</i> interaction. The arrows indicate the VapB and VapC bands. M - Molecular Weight Marker (kDa). Pull-down assay was perfomed more than 5 times. (<b>B</b>) Ligand affinity blotting. To analyze specific binding between VapB and VapC, the VapC and LipL32 proteins (negative control) were subjected to 15% SDS-PAGE (left panel) and transferred to nitrocellulose membrane (right panel). After blocking, the membrane was incubated with a VapB solution (3 µg ml⁻¹). Following extensive washing, the membrane was incubated with anti-VapB antibodies. M - Prestained Molecular Weight Marker (kDa). VapB in solution bound to both monomeric (15.1 kDa) and dimeric (30.2 kDa) forms of VapC immobilized in the membrane, denoting <i>in vitro</i> interaction. VapB did not bound the negative control, protein LipL32.(<b>C</b>) Western blot control showing that anti-VapB antibodies recognizes specifically VapB, and not VapC.