Supplementary Materials and Methods
APC/C In vitro ubiquitination assay and in vitro inhibitory assay
In vitro ubiquitination assay and APC/C inhibitory assay were performed as
previously described (Braunstein et al., 2007; Reddy et al., 2007). For the in vitro
ubiquitination of RASSF1A, HeLa cells arrested at G1/S boundary, mitosis or G1
phase were lysed with lysis buffer (50mM Tris (pH 7.5), 250mM NaCl, 0.1% Triton
X-100, 1mM EDTA, 1mM DTT and phosphatase and protease inhibitor (Roche,
Indianapolis, IN)). APC/C was immunoprecipitated from the cell lysates by
anti-Cdc27 antibody and Protein-G sepharose (Invitrogen). In vitro ubiquitination was
carried out in 10μL reaction containing 1X reaction buffer (40mM Tris (pH 7.6),
1mg/ml bovine serum albumin, 1mM DTT, 5mM MgCl2), 1X energy regeneration
mix (10mM phosphocreatine, 50mg/ml creatine phosphokinase, 0.5mM ATP), 50
mM ubiquitin, 1 mM ubiquitin aldehyde, 1pmol E1, 5pmol UBcH10, 1mM okadaic
acid and 1μL of
35
S-labeled TnT quick coupled In vitro-transcribed/translated
RASSF1A (Promega, Madison, WI). Where indicated, 80ng of recombinant Aurora A
or Aurora B (Cell Signaling Technology, Danvers, MA) was added to the reactions.
The reactions were incubated at 37oC for 30 minutes and analyzed by SDS-PAGE and
autoradiography.
For the APC/C inhibitory assay, APC/C immunoprecipitated from mitotic HeLa
cells were washed with Buffer A (50 mM Tris·HCl, pH 7.2, 1 mM DTT, 10% glycerol,
1 mg/ml BSA), followed by washing with Buffer A containing 0.3M KCl for 10
minutes at 4 oC twice, and finally washed with Buffer A. Cdc20(WT) and Cdc20(7A)
were produced by in vitro-transcription/translation reaction with unlabeled
L-methionine. The
35
S-labeled N-terminal Cyclin B1 fragment was generated by in
vitro-transcription/translation reaction with
35
S-methionine. The inhibitory reaction
mixture in 10μL containing 40mM Tris, pH 7.6, 1mg/ml bovine serum albumin, 1mM
DTT, 5mM MgCl2, 10mM phosphocreatine, 5 mg/ml creatine phosphokinase, 0.5mM
ATP, 50mM ubiquitin, 1mM ubiquitin aldehyde, 1pmol human E1, 5pmol UbcH10, 1
mM okadaic acid, APC/C beads, 0.6μL In vitro-translated Cdc20, and 4μM
MBP-RASSF1A or 1.2μL in vitro-translated RASSF1A was first incubated at 4oC for
20 minutes. Then, the reaction was started by adding 1μL
35
S-cyclin B1. Where
indicated, 80ng of Aurora A or Aurora B was added to the reaction mixture. The
reaction was incubated at 30oC for 45 minutes and analyzed by SDS-PAGE and
autoradiography. The intensity of poly-ubiquitinated Cyclin B1 (Cyclin B1poly(ub)) was
quantified by Quantity One software (Biorad).
In vitro kinase assay
Aurora A or Aurora B kinase assay was performed according to manufacturer’s
protocol (Cell Signaling Techonology). Reactions were set up in 10μL consisting of
80ng of Aurora A or Aurora B and 100ng of purified MBP-RASSF1A in 1X reaction
buffer (25mM Tris (pH 7.5), 5mM beta-glycerophosphate, 2mM DTT, 0.1mM
Na3VO4 and 10mM MgCl2) and 32P-γ-ATP. The reactions were incubated at 30oC for
20 minutes and analyzed by SDS-PAGE and autoradiography.