1. Volume 11, Issue 17, Pages 1347-1352 (September 2001)
The anaphase inhibitor Pds1 binds to the APC/C-associated protein Cdc20 in a destruction box-dependent manner 
Zoe Hilioti, Yin-Shin Chung, Yuko Mochizuki, Christopher F.J Hardy, Orna Cohen-Fix 
Current Biology 
Volume 11, Issue 17, Pages (September 2001)
DOI: /S (01)

2. Figure 1
The two-hybrid interaction between Pds1 and Cdc20 is dependent on Pds1's destruction box. (a) The Cdc20 and Pds1 constructs used in the two-hybrid screen. The Cdc20 fragment isolated by the two-hybrid interaction contained the WD-repeats (hatched bars) but lacked the two destruction boxes (solid bars). The dependence on the Pds1 destruction box was tested by using an N-terminal fragment of Pds1 (amino acids 1–174), with or without a functional destruction box (N-Pds1 and N-Pds1Δdb, respectively). The destruction box mutation was generated by replacing the RLPL amino acid sequence with amino acids ALPA, as described previously [7]. Note that the two hybrid interaction between Pds1 and full-length Cdc20 could not be tested because cells expressing full-length CDC20 fused to the GAL4 activation domain (AD) grew extremely slowly (data not shown), possibly due to the toxic effect of elevated Cdc20 levels [2, 3]. (b) Growth of the indicated two-hybrid combinations on plates lacking tryptophane and leucine, to select for the bait and prey plasmids, respectively, or on a plate lacking tryptophane, leucine, histidine, and adenine to test for the two-hybrid interaction. (c) Western blot analysis using anti-Pds1 antibodies to detect the GAL4 binding domain (BD) fusions to Pds1, N-Pds1, and N-Pds1Δdb in the two-hybrid tester strain. (d) The β-galactosidase activity of the two-hybrid tester strain expressing AD-Cdc20 and the Pds1 derivatives, as indicated
Current Biology  , DOI: ( /S (01) )

3. Figure 2
Pds1 interacts in vitro with Cdc20 but not with Cdh1. (a) GST-Pds1 and GST-Pds1Δdb fusions were expressed in E. coli and bound to glutathione agarose. GST (lanes 3 and 7), GST-Pds1 (lanes 1 and 5), and GST-Pds1-Δdb (lanes 2 and 6) were incubated with Cdc20 (a) or Cdh1 (b) that had been expressed in reticulocyte lysate in the presence of [35S]methionine. After 2 hr of incubation, the beads were washed extensively, and the bound proteins were resolved by SDS-PAGE and analyzed by autoradiography. Fifty percent of the bound protein was loaded. The input lanes contain 3% or 5% of the Cdc20 or Cdh1 lysates, respectively, added to each incubation (lanes 4 and 8). The Δdb mutation removed the RLPL destruction box element and replaced it with leucine and glutamic acid, as described [7]. (b) Testing a two-hybrid interaction between Pds1 and Cdh1. See Figure 1 for details. The Pds1 and Cdc20 constructs were as described in Figure 1 and served here as a control
Current Biology  , DOI: ( /S (01) )

4. Figure 3
Pds1 interacts with Cdc20 in vivo, and this interaction is not abolished when the mitotic checkpoint is activated. (a) Early log phase cultures of cdc mutant strains expressing Pds1-HA and Cdc20-Myc (strain ZH2601-9A) or Cdc20-Myc alone (strain ZH2601-3C) were shifted to the nonpermissive temperature (37°C) for 5 hr and then harvested. Extracts made from these cells were subjected to immunoprecipitation (IP) using anti-HA antibodies coupled to beads as described under Materials and methods. Total extracts and eluted fractions were subjected to SDS-PAGE and analyzed by Western blot using anti-HA antibodies (top panels) or anti-Myc antibodies (bottom panels). (b) Strains (Pds1-HA) and (Pds1-HA GAL-Pds1Δdb) [22] were arrested in S phase with 0.2 M hydroxyurea. In all cases, cells were treated for 30 min with galactose prior to the release from the S phase arrest; low-level Pds1Δdb was achieved by releasing the cells into media containing glucose whereas high levels of Pds1Δdb were generated by releasing the cell into media containing galactose. The levels of β-tubulin are shown as a loading control. By cell morphology (data not shown), the wild-type strain (1521.0), which progressed through the cell cycle, entered a new cell cycle at around 90 min (glucose) or 120 min (galactose). As a result, the reaccumulation of Pds1-HA can be observed. (c) The strains described in panel (a) were prearrested in S phase with hydroxyurea, after which they were released from the arrest at the nonpermissive temperature in the absence or presence of nocodazole. Two hours after the release cells were harvested and processed as described in (a). The left and the right panels show the IP results in the absence and presence of nocodazole, respectively
Current Biology  , DOI: ( /S (01) )