in TTBS buffer (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.

1% Tween-20) with 4% ski

m milk and then incubated with the indicated antibodies. Finally, membranes were
washed, incubated with HRP-conjugated secondary antibodies, and developed with
ECL (Thermo Scientific).
GST pull down
1 g of GST or the relevant GST-fusion protein was incubated with recombinant prot
eins for 1 hour in IPP-150 buffer (10 mM Tris-HCl, 150 mM NaCl and 0.1% NP-40) a
nd 20 L of GST beads were added and incubated at 4C for 90 minutes with rotation.
For mass spectrometry assay, 10 g of GST or GST-MEKK2 was embedded into a column
using GST beads and then 2 mg of cell lysates from WNT 10b expressing ST-2 osteo
blsats were added for at 4C for overnight. Beads were centrifuged and washed 3 ti
mes and eluted with IPP-325 buffer (325 mM NaCl, .1% NP-40, 10 mM Tris-HCl) cont
aining glutathione.
Pulse-chase assay
HEK293T cells were maintained at 37C, 5% CO2. One day before transfection, cells
were plated in a 6 well plate with 4.2 x 105 cells/ml. After 48 hours of transfe
ction, cells were changed to starvation media (Cys/Met free DMEM, 3% dialyzed FB
S, Gln, HEPES) and incubated 1 hour at 37C, 5% CO2. Then, cells were harvested an
d resuspended with 2 ml starvation media with 1 mCi 35S (Perkin Elmer) and incub
ated for one hour on rocking stand at 37C, 5% CO2. After incubation, cells were w
ashed with PBS and centrifuged with 1300 x g, 5 min. Medium was changed to chase
media (DMEM, 5% FBS, +Cys, +Met) and incubated on a rocking stand for 0, 3, 6,
9 hours at 37C, 5% CO2. After incubation, cells were harvested and immunoprecipit
ated with Flag M2 beads (sigma) and eluted proteins were detected with autoradio
graphy.
In vitro ubiquitination/deubiquitination assay
500 nM UbcH3 (Millipore), 0.1 g SCF?-TrCP1 (activated, Millipore), 100 nM ?-caten
in (Millipore), 2 M ubiquitin was mixed in ubiquitination buffer containing 25 mM
MOPS, pH 7.5, 0.01% Tween20, 5 mM MgCl2, 2 mM ATP and 1 mM DTT. Phosphorylation
was initiated by adding 100 nM MEKK2 (Invitrogen) and incubating for 1 h at 30?
C. Afterwards, 10 nM E1 (Millipore) was added to initiate ubiquitination, and th
is was incubated for 1 h at 37?C. Then, 5 mM EDTA was added for 15 min at room t
emperature to stop the ubiquitination reaction. Finally, 400 nM USP15 (ENZO Life
Sciences) was added for deubiquitination and incubated for 1 h at 37?C. SDS sam
ple buffer was added to stop the reaction.
Luciferase Reporter assay
C3H10T1/2 osteoblasts grown on 12-well plates were transiently transfected using
Effectene (QIAGEN) with 0.5 g of TOP-FLASH plasmid and 50 ng of a plasmid expres
sing Renill luciferase (Promega). 12 hours after transfection, 100 ng/ml of Wnt3
a (R&D systems) in serum-free media was added for 24 hours. For Wnt-signaling in
hibition, 1 g/ml DKK1 (R&D systems) was added one hour prior to Wnt3a treatment.
Normalized luciferase activity was obtained using the Dual-Luciferase Reporter a
ssay System (Promega).
Statistics
Where applicable, statistical analysis was performed by first assessing for norm
ality using a Shapiro-Wilk test and equality of variances using a F-test followe
d by a 2-tailed, unpaired Student s t-test. Where multiple comparisons were perfor
med, a one-way or two-way ANOVA with Bonferroni-corrected Student t-tests as pos
ttests were performed. A P value less than 0.05 was considered significant. *P<0
.05, **P<0.01, ***P<0.001. In general, animal experiments were powered to detect
a 20% difference in BV/TV assuming a standard deviation of 15% of the mean with
an a of 0.05 and a of 0.80. No animals or other experimental samples were exclu
ded from analysis. Except where otherwise indicated, experiments were repeated 3
times. All images shown are representative.