ß-gal NME2 NME1 Actin NME1 NME2 Scrambled RISC Free 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 MDMX Gankyrin p53 MTBP Rb Actin Lys 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 NME 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 NME2 Long exp. NME1 Long 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 Ab 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, ) Polanski and Maguire et al, Supplementary data Figure 3 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. Scrambled NME2 MDM2 NME2+MDM2 Scrambled NME2 MDM2 NME2+MDM2 MDM2 NME2 Actin MDM2 NME2 Actin B A