FIGURE 5.

Vpr binds to hit compound 3 and vipirinin in a competitive manner. All bands are FLAG-tagged Vpr proteins bound to compound-immobilized beads. A, Vpr in yeast total cell lysates (2 μg in 500 μl of binding buffer) was preincubated in binding buffer containing vipirinin (+) or DMSO (−). Precipitants were allowed to settle, and soluble fractions were carefully removed and incubated with 10 μl of 3- or vipirinin-immobilized beads. Competition was observed when increasing amounts of vipirinin were added. B, Vpr mutant S28A does not bind to either 3- or vipirinin-immobilized beads. No chemiluminescent bands were detected despite prolonged exposure times. All bound proteins were detected by Western blotting after SDS-PAGE. V, vipirinin.