(A) Lsb5 was purified and incubated with GST–Arf3 beads with Arf3 in either the active (Q71L) or inactive (T31N) form as described in the Materials and methods section. Binding was observed only with the active Arf3p and Lsb5p. (B) Lsb5p was then incubated with ubiquitin–agarose or, as a control, IgG–agarose beads. After washing, binding was assessed by SDS/PAGE. Binding was detected with Lsb5 only on the ubiquitin-conjugated beads. I, input; FT, flow through; W1, first wash; W3, third wash; B, bound; Ubi, ubiquitin; Ag, agarose.