Immobilized Ddi1 and Rpn1 reconstitute SCF^Ufo1 complexes <i>in vitro</i>.

<p>A. ^GSTDdi1 or control GST on GSH beads were incubated with yeast extract from cells that produced ^mycCdc53 and ^GFPUfo1. Analysis was by Western blotting with anti-myc, anti-GFP, and anti-GST antibodies. T represents 10% of the yeast extract with which the beads were incubated. B.^GSTRpn1, ^GSTRpn10, or GST beads were incubated with yeast extract from cells that produced ^mycCdc53 and ^GFPHo mixed with bacterial lysate with recombinant ^HISUfo1. The bead fraction was analysed by Western blotting with anti-myc, anti-HIS, anti-GFP, anti-Ddi1, and anti-GST antibodies. T represents 10% of the yeast extract incubated with the beads. C. ^GSTUfo1 WD40 domain, ^GSTCdc53, or control GST on GSH beads were incubated with bacterial lysate from cells that produced recombinant ^HISΔΔDdi1. The bead fraction was analysed by Western blotting with anti-HIS and with anti-GST antibodies as indicated. T is 10% of the ^HISΔΔDdi1 bacterial lysate incubated with the beads. D. The ^HISΔΔDdi1 bacterial lysate was incubated with ^GSTRpn1, ^GSTRpn10, or GST beads and analysed as above.