ß-gal

NME2

NME1

Actin

NM

E1

NM

E2

Scra

mbl

edR

ISC

Fre

e

Supplementary data Figure 1B.

Antibody L-15 is specific for NME2

and sc-465 is specific for NME1.

Western blot of MCF-7 cells treated

with the indicated siRNA for 48

hours and probed with the indicated

antibodies.

A

Gankyrin

p53

MTBP

Rb

Actin

Lys 4 5 6 7 8 9

MDMX

Gankyrin

p53

MTBP

Rb

Actin

Lys 4 5 6 7 8 9 Supplementary data Figure 1A.

Fractions from the column analysed

in main Figure 1 were analysed by

western blotting with the indicated

antibodies. Lys indicates 50μg

whole cell lysate from HEK293 cells.

Note that the distribution of MDM2

binding proteins differs such that

some preferentially bind MDM2 in

fraction 5 and others in fraction 6.

Polanski and Maguire et al, Supplementary data Figure 1

B

MG132

*NME1 long form

NME2-K12Q

NME2

MDM2

Actin

β-gal

GFP

NME2

Actin for NME2

++++++

+ +

+

+

++

------

-----

-

-----

------

MDM2 long exposure

MDM2 short exposure

Polanski and Maguire et al, Supplementary data Figure 2

Supplementary data Figure 2. NME2 decreases the steady-state level of MDM2 in a kinase

independent manner that is inhibited by the proteasome inhibitor MG132. Inhibition of the 26S

proteasome rescues the NME2-mediated decrease of the levels of MDM2 independent of the kinase

status of NME2. The panel shows the results of western blot analysis with the indicated antibodies of

protein lysates from H1299 cells transfected with constructs expressing MDM2 (0.5μg), 3μg of either

NME1, NME2 or NME2-K12Q as indicated, and 0.3μg of GFP. For NME1 antibody is sc-465, for

NME2 antibody is L-15. Cells were seeded into six-well plates and incubated for 24h prior to

transfection. 19h after transfection, some samples as indicated were treated with the proteasome

inhibitor MG132 at 10μM for 5h prior to harvesting. *Note that the long form of NME1 is generated

from an artificial Kozak sequence which promotes efficient use of an upstream in-frame ATG that is

otherwise not utilised efficiently. This adds 25 amino acids allowing for clear discrimination between

endogenous and transfected NME1 forms.

NME1

Actin for NME1

NME2Long exp.

NME1Long exp.

A. Western blot analysis of 117 cells transfected with the indicated siRNA confirms

migration differences between NME1 and NME2. Western blot with pan NME1/2 antibody

Ab31019. Note that NME1 and 2 migrate at distinct rates as confirmed in B. Western blot

analysis of p53-/-, MDM2 -/- mouse embryo fibroblasts transfected with plasmids

expressing either human NME1 or NME2 as indicated and probed with the indicated

antibodies. We used MEFs to try to avoid potentially confounding effects from

endogenous human NME1 or 2. MEFs were kindly provided by Prof. S . Jones (Jones,

S.N., Roe, A.E., Donehower, L.A. and Bradley, A. (1995) Rescue of embryonic lethality in

Mdm2-deficient mice by absence of p53. Nature, 378, 206-8.)

Polanski and Maguire et al, Supplementary data Figure 3

Actin

NME

siRNA

Human NME1Human NME2

β-actin

GFP

NME1NME2 --

--+

++

+- -

B

A

Actin

GFP

Polanski and Maguire et al, Supplementary data Figure 4

Supplementary data Figure 4. Analysis of cell motility following siRNA transfection

with additional siRNAs confirms results presented in Figure 6. Cells were treated as

per Figure 6, but with different siRNAs for NME2 #7 and MDM2 #9. Panels in B show

western blot analysis of the samples in A transfected with the indicated siRNAs and

analysed with the indicated antibodies ; IF2 used for MDM2, Ab31019 for NME1/2 and C-

2 for actin.

Scram

bled

NME2

MDM

2NM

E2+M

DM2

Scram

bled

NME2

MDM

2NM

E2+M

DM2

MDM2

NME2

Actin

MDM2

NME2

Actin

117 1.27B

A