Supplemental Figure 1
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Fig. S1.
Expression of proteins involved in Wnt-regulated b–catenin targeting complex in different cells. To
permit the direct comparison of protein complexes in different cell lines, the
expression level of proteins known to associate with APC was determined.Equal amounts of protein from cell
lysates of the indicated cell lines were probed with antibodies to the proteins
as shown.The relevant band for
Axin-1 and 2 are marked with stars on the right of the blots.HeLa cells expressed the least APC and
also had comparatively low level of b-catenin.HCT 116 cells expressed higher levels of APC and b-catenin.The
latter is due to a mutation
inb-catenin
that eliminates the phosphorylation site (serine 45) required for its
ubiquitination.MDCK cells also
contained high levels of APC and b-catenin,
consistent with the fact that MDCK cells form extensive cell-cell contacts,
which require b-catenin.In cell lines expressing
only truncated APC (namely SW480,
HT-29 and DLD-1), the levels of truncated APC protein and of b-catenin were high.It is possible that failure of b-catenin
degradation due to mutations in either APC or b-catenin
itself, leads cells to compensate by
up-regulating proteins that normally help to control b-catenin, such as APC and
Axin.The up-regulation of
Axin-2/conductin by b-catenin-TCF-mediated transcriptional regulation
has already been confirmed (Jho et
al., 2002; Lustig and Behrens, 2003).Indeed, we detected higher levels of Axin-2/conductin in tumour
cell lines with mutant APC or b-catenin
when compared to HeLa cells
(Figure 1). We could not detect Axin2 in MDCK cells, possibly because the
antibody we used was specific for human Axin-2 and failed to detect canine
Axin-2.The levels of GSK3band PP2A-C did
not vary greatly between cell lines. The B56 subunit of PP2A was slightly
increased in cells expressing full length APC compared to the three colon
cancer cell lines with truncated APC.
Supplemental Figure 2
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Fig. S2.Subcellular fractionation of HeLa cells
proteins. Control or SB-treated (12 hours) HeLa (A), DLD-1 (B) and SW480 (C)
cells were fractionated into cytosol, membrane, nuclear, and insoluble/cytoskeletal
pools using Compartmental Protein Extraction Kit from Chemicon (Cat No. 2145)
as per the manufacturer’s instructions.Each fraction was probed with antibodies against APC, and Topoisomerase
II (to mark nuclear protein pools), b-catenin,
GSK3b, and GAPDH (to
confirm cytosolic fractions).(A)
In HeLa cells, APC fractionated predominantly into cytosolic and nuclear
pools.Treatment with SB reduced
the cytosolic amount of APC but did not affect the relative distribution of the
other proteins.The increased b-catenin protein became
detectable in cytosolic and nuclear fraction after SB treatment most likely due
to its increased levels.GSK3b was detectable in all fractions
and showed an intriguing migration pattern that was specific to the insoluble/cytoskeletal
fraction.(B) APC was mostly
cytosolic in DLD-1 cells but could also be detected in the other
fractions.(C) In SW480 cells,
there was more APC in the insoluble/cytoskeletal pool and relatively less in
the nuclear and member fractions.In either case the distribution of APC did not respond to the inhibition
of GSK3b.In SW480 and DLD-1 cells, Topoisomerase
II was detected strongly in the insoluble fraction indicating that this method
may not have solubilised nuclear proteins efficiently in these cells.(D) Because the cytosolic fractions
prepared by the above fractionation method yielded samples that were too dilute
to allow detection of APC after further separation on glycerol gradient fractionation
and because SB treatment resulted in some shift of APC to nuclear fractions, we
needed to compare how our solubilisation affected APC when related to these
marker proteins.So we tested the
distribution of APC between the soluble and insoluble material using our lysis
conditions.Cell lysates were
prepared as described in the Materials and Methods and soluble material and the
insoluble material were probed with antibodies to APC, Topo II, and GAPDH.These data show that the distribution
of APC between the soluble material, which is what was applied to our glycerol
gradients, and the nuclear material which was removed prior to fractionation
did not change significantly by treatment with SB.
Supplemental Figure 3
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Fig. S3.
Inhibiting GSK3b
leads to a small molecular weight shift that can be eliminated by l-phosphatase. Cell lysates from
non-treated (Non) or SB-treated (SB) cells were treated with control buffer
(Ctrl) or the same buffer containing l-phosphatase
(l) prior to PAGE and
immunblotting with anti-APC antibodies.Treatment with phosphatase produced a molecular weight shift that was
slightly larger in the control cells consistent with the idea that inhibiting
GSK3b affects the
phosphorylation of APC in cells.
Supplemental Figure 4
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Fig. S4. The total
amount of Axin-1 does not change in HeLa cells after SB treatment. Equal amount
of proteins from cell lysates of control or SB treated HeLa cells were probed
with antibodies against Axin1 to show that the total amount of this protein is
not affected by the treatment. Please note that the reason for the difference
in the appearance of the blot between this Figure and Figure 1 is a change in
the antibody provided by the company from which we purchases the reagent.
