SUPPLEMENTAL DATA FIGURE LEGENDS FIGURE S1 MAD3PMKB1

1 SUPPLEMENTAL TABLE THE EMPYREAN STUDY COMMITTEES PRINCIPAL INVESTIGATOR
11 SUPPLEMENTAL FIGURES SF1 SAMPLES CLUSTER BY STRAIN PLOT
2170463 §217046—ELIGIBILITY FOR SUPPLEMENTAL EDUCATIONAL ASSISTANCE 2170463 §217046 ELIGIBILITY

3 SUPPLEMENTAL TABLE 1 PARTIAL CORRELATIONS† BETWEEN DIETARY FACTORS‡
7 SUPPLEMENTAL INFORMATION VAKIFAHMETOGLUNORBERG ET AL SUPPLEMENTAL INFORMATION SUPPLEMENTAL
7 TABLE 1 SUPPLEMENTAL INFORMATION ON THE ANALYZED STUDIES

Supplemental Data

Supplemental Data

Figure Legends

Figure S1. Mad3p-mkb1 does not associate with Cdc20p following checkpoint arrest induced by Mps1p overexpression. myc-Mad3p wild-type and mkb1 were immunoprecipitated with anti-Mad3p antibodies from strains overexpressing myc-Mps1p (YJB900 and YJB901). Mad3p and the co-immunoprecipitating Cdc20p were detected by immunoblotting. No Cdc20p or Mad3p were detected in immunoprecipitates from an extract lacking Mad3p (YJB657).


Figure S2. Mad3p-mkb1 can associate with Bub3p.

(A) Wild-type (YJB815) or mkb1 myc-Mad3p (YJB818) was immunoprecipitated from cycling or benomyl-treated cells using anti-myc antibodies and the levels of co-immunoprecipitating Cdc20p or Bub3p-HA were detected by immunoblotting with anti-Cdc20p or anti-HA antibodies, respectively. myc-Mad3p in the immunoprecipitates was detected with anti-myc antibodies. Neither Cdc20p, Mad3p nor Bub3p was detected in immunoprecipitates from a strain lacking a myc tag (YJB812).

(B) Using the same strains as in (A), Bub3p-HA was immunoprecipitated and the levels of co-immunoprecipitating Mad3p and Mad3p-mkb1 were detected by immunoblotting with anti-myc antibodies. No Mad3p co-immunoprecipitated from a strain lacking an HA tag (YJB697).


Figure S3. KB1 is important for Cdc20p instability.

(A) myc-Mad3p wild-type (YJB800), mkb1 (YJB801) and mad3 (YJB793) strains were arrested in S, M, or M plus benomyl. Samples were taken at the indicated times after cycloheximide addition and Cdc20p levels were monitored by immunoblotting using anti-Cdc20p antibodies.

(B) myc-Mad3p wild-type (YJB800) or mkb2 (YJB924) expressing strains were arrested in M-phase with checkpoint activation by overexpression of Pds1p-mdb with benomyl addition. Samples were taken at the indicated times after cycloheximide addition and Cdc20p levels were monitored as in (A).

(C) Cdc20p stability in other checkpoint mutants. Wild-type (YJB759), mad1(YJB768), bub1 (YJB769) and mad2 (YJB906) strains were arrested in M-phase by Pds1p-mdb expression. Cdc20p levels were monitored following cycloheximide addition as in (A).


Figure S4. Wild-type and mkb1 Mad3p were purified from baculovirus-infected cells and incubated with control beads (lanes 1 and 3) or with Cdc20p beads (lanes 2 and 4). Twenty-five percent of the input Mad3p is shown in the upper right hand panel. Cdc20p and Mad3p were detected by immunoblotting with anti-Cdc20p and anti-Mad3p antibodies.


Figure S5. The Mad3p KEN box peptide and Hsl1p D-box and KEN box peptides can compete for APC-substrate binding to Cdh1p. Cdh1p beads were pre-incubated with 500 µM of the indicated peptides prior to incubation with MBP-Clb2p or MBP-Hsl1p. The beads were washed and bound proteins detected by immunoblotting with anti-MBP antibodies. His6-Cdh1p peptide competition assays were performed using 5 l of His6-Cdh1p beads in 0.1 ml of the same buffer as was used for the Cdc20p binding reactions. Peptides (500 M) were pre-incubated with the Cdh1p beads for 30 minutes followed by addition of 0.5 g of either wild-type MBP-Hsl1p or MBP-Clb2p and an additional 30 minute incubation at 4oC.


Figure S6. Mad3p associates with the APC in M-phase and under checkpoint activation conditions. Wild-type (YJB800) or mkb1 (YJB801) Mad3p was immunoprecipitated with anti-Mad3p antibodies in M-phase or under checkpoint activation conditions and co-immunoprecipitated Cdc16p (an APC subunit) and Cdc20p were monitored by immunoblotting with anti-HA and anti-Cdc20p antibodies, respectively. Unlike Mad3p, Mad3p-mkb1 does not interact with either Cdc20p or the APC.


Achievement Goals 7 Supplemental Materials in Their own Words
ANNEX II SUPPLEMENTAL STATUTORY DECLARATION IN THE MATTER
ANNOUNCEMENT REQUEST FOR PROPOSAL GROUP LIFEDISABILITYSUPPLEMENTAL LIFE DECEMBER 2017


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