Supplementary Figure 5: Specificity of anti-Integrase intrabodies.

(a) Yeast growth on histidine-lacking plates (with the addition of appropriate 3AT concentration, a His3 inhibitor) demonstrates interaction specificities for 112A and 12X intrabodies. In particular, it is shown how the splitting of ScFvs in its constituent Variable domains abolishes its cross-reactivity with P300mut for both VH-112A and VH-12X. The panel a illustrates VH-112A to be a specific anti-acetyl-Integrase intrabody, and VH-12X to target Integrase regardless of its acetylation status. (b) Yeasts are restreaked from transformation plates on fresh selective plates. The effect of splitting ScFv-112A variable domains is consistently evident. (c) Beta-galactosidase lift assays performed on restreaked colonies carrying 112A/Integrase prey/bait pairs further confirm the specific acetyl-Integrase binding of VH-112A, compared to its ScFv version. (d) Cross-reaction between P300mut bait and anti-Integrase intrabodies (ScFv-112A and ScFv-12X) is totally suppressed by a low dose of 3AT (1.25mM), which is the same that was used to suppress transactivation of P300wt bait (see Supplementary Figure 3b). The panel shows three independent clones of ScFv-112A and ScFv-12X restreaked from the transformation plate.