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. Author manuscript; available in PMC: 2016 Jan 15.
Published in final edited form as: Dev Cell. 2015 Feb 9;32(3):358–372. doi: 10.1016/j.devcel.2015.01.003

Figure 2. The ABBA motif binds to CDC20 between blades 2 and 3 of the WD40 domain.

Figure 2

(A) Immobilized peptides containing wild-type (wt) and mutant (mut) ABBA motif from Cyclin A2, BubR1 and Bub1 were incubated with extracts from prometaphase (PM) and metaphase (M) HeLa cells (see Experimental Procedures). Input signals and background binding to beads are shown. For the input, 1/30 of the extract used for the pulldown was loaded. Actin is the loading control for the input.

(B) Mean and Standard error of the Mean (SEM) from 4 independent experiments as shown in (A). For each peptide the binding is normalised to the binding of the wt peptide in PM.

(C) Sf9 cell extracts expressing Hs CDC20 were incubated with the indicated biotinylated peptides and a 200-fold excess of a competing non-biotinylated peptide followed by purification with streptavidin beads. See also supplemental Fig. 2.

(D) Bar charts show mean and SEM from 3 independent experiments normalized to CDC20 binding after competition with the respective Cyclin A or BubR1 mutant peptide. The competition of wild type Cyclin A and BubR1 peptides to the respective mutant peptide was analysed using a two-tailed paired t-test (*, p<0.05; **, p<0.005; ***, p<0.0005; n.s., not significant).

(E) Streptavidin beads bound to SBP-CDC20 were incubated with GST, GST-Cyclin A2 wt or ABBA mutant. Proteins retained on the beads were analysed by quantitative immunoblotting and normalized values for Cyclin A2 are shown. Results are representative of 2 experiments.

(F) Streptavidin beads were incubated with insect cell lysate expressing SBP-BubR1, unbound proteins washed away, and beads incubated with cell lysate expressing untagged CDC20 with GST, or GST-Cyclin A2 wt or ABBA mutant. Proteins retained on the beads were analysed by quantitative immunoblotting.

(G) Streptavidin beads with rMCC bound via SBP-BubR1 were incubated with GST, GST-Cyclin A2 wt or ABBA mutant. Proteins retained on the beads and in the flow-through (FT) were analysed by quantitative immunoblotting and normalized values for Cyclin A2 are shown. Results representative of 2 experiments.

(H) Peptide pulldown using immobilised peptides and total extract from siRNA CDC20-depleted prometaphase cells expressing Venus-Flag, or siRNA resistant Flag-CDC20 wild-type or Y279E/I280Q mutant. Actin is a loading control.

(I) Mean and SEM from at least 3 independent experiments shown in (H). For each peptide the binding is normalised to the binding of wt CDC20 and values are normalised to input. Paired t-test was used for statistical analysis (*, p<0.05; **, p<0.005; n.s., not significant).