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Supplementary Figure Legends
Supplementary Figure 1
A. Top Predominant p53 ubiquitination in the cytoplasm correlates with differential p53
stability in cytoplasm and nucleus of unstressed cells. p53 has a much shorter half-life in
the cytoplasm (20 min), compared to the nucleus (60 min). RKO cells where treated with
cycloheximide (CHX) for the indicated times, followed by fractionation and
immunoblotting for p53. Bottom Densitometry of immunoblot shown on the left. Hsp90
and HDAC as purity controls of cytoplasmic and nuclear fractions, respectively.
B. Ubiquitination of ectopically expressed p53 occurs predominantly in the cytoplasm,
even after nuclear export block induced by LMB treatment. H1299 (p53-/-) cells were
transfected with p53 and MDM2, treated with LMB where indicated and fractionated.
Equal levels of non-ubiquitinated p53 were loaded to assess p53 ubiquitination.
C. To exclude the possibility that HAUSP is more active in the nucleus than in the
cytoplasm, we utilized stable LS126 cells (human colon cancer cells expressing wt p53)
with Dox-inducible HAUSP shRNA 10. LS126 cells were grown in the absence or
presence of doxycycline. p53 from nuclear and cytoplasmic fractions was normalized for
equal amounts of non-ubiquitinated p53 and blotted as indicated. Note that Ub-p53 after
HAUSP silencing does not accumulate in the nucleus, indicating that the lack of nuclear
Ub-p53 is not due to enhanced de-ubiquitination by nuclear HAUSP.
D. To exclude the possible that ALLN is not sufficient to block the degradation of all
nuclear p53, we performed subcellular fractionation experiments in the presence of a
cocktail of the classic proteasome inhibitors ALLN + MG132. Results are identical to
using ALLN alone (see Figure 1F). RKO cells were treated with ALLN + MG132 for 3
hrs, followed by fractionation. Nuclear and cytoplasmic fractions normalized for equal
total protein (left) or equal amounts of non-ubiquitinated p53 (right) were
immunoblotted. Hsp90 and HDAC as markers for cytoplasmic and nuclear fractions.
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Supplementary Figure 2
A. The p53 9R mutant (all Lysines to Arginines in NLS I-III) is ubiquitination-defective.
Either wild-type p53 or the 5R and 9R mutants (see Figure 4A) were transfected into
H1299 cells along with MDM2 as indicated. After 24 hours cells were subject to nuclear
and cytoplasmic fractionation and analyzed by immunoblotting for p53. Loadings were
normalized for equal amounts of non-ubiquitinated p53.
B. The p53 3R (all lysines to arginines in NLS I) and 6R mutants (all lysines to arginines
in NLS II-III) are ubiquitination-defective. Either wild-type p53 or the 3R and 6R
mutants were transfected into H1299 cells along with MDM2 and treated or left untreated
with proteasome inhibitor ALLN as indicated. After 24 hours cells were analyzed by
immunoblotting for p53. Loadings were normalized for equal amounts of nonubiquitinated p53.
Supplementary Figure 3
We used 4 nM LMB in all our experiments, since it is the concentration routinely used in
the literature to block nuclear export of p53 5. To further ensure that the observed
difference in the kinetics of nuclear p53 accumulation after DNA damage stress versus
export blockade is not due to suboptimal LMB concentrations (Figure 6A), we ran an
additional control experiment in the presence of 15 nM LMB. We find that even 4-fold
higher LMB concentration does not accelerate nuclear p53 accumulation (compare to
Figure 6A). Thus, 4 nM LMB is sufficient to completely block nuclear export.