Supplementary information

Materials and methods

Expression and purification of GST-tagged proteins

The GST-NES1, GST-NES2 and GST expressing plasmids were transformed separately into E. coli BL21 (DE3) pLysS. Transformed cells were inoculated into 100 ml of LB medium and incubated at 37ºC with vigorous shaking until an A600 value of 0.6 was reached. Growth of the E. coli was induced by the addition of 1 mM isopropyl 1-thio--D-galactopyranoside (IPTG). After 4h cultures were harvested. The cells were lysed with a sonicator and centrifuged at 35,000 rpm for 20 min to remove cell debris. The supernatants were loaded onto a Glutathione Sepharose 4B column

(MicroSpin GST Purification Module; GE HEALTHCARE), and bound GST-tagged proteins were eluted in elution buffer (50 mM Tris-HCl pH 8.0, 10 mM reduced glutathione). An aliquot of each eluate was analyzed using SDS-PAGE and the fractions containing the protein were quickly frozen in small aliquots using liquid nitrogen.

Two-dimensional electrophoresis

Protein separation by 2D-PAGE was as follows: extracts containing 5 g of protein complexes were re-suspended in 8 M urea, 2 M thiourea, 2% (v/v) Triton X-100, 420 mM 2-mercaptoethanol, and

2% pH 3–10 IPG buffer, and loaded onto Immobiline Dry strips (IPG strip; Amersham) in the pH range 3–10. Gels underwent active rehydration at 500 V for 1.5h, followed by a further 4.5h focusing with a total of 10,500 V applied. Following IEF, gels were equilibrated in reducing buffer

(50 mM Tris–HCl, 1 M urea, 2% (w/v) SDS, 30% (v/v) glycerol, and 2% (w/v) DTT, pH 6.8) for 30 min followed by equilibration in alkylation buffer (50 mM Tris–HCl, 6 M urea, 2% (w/v) SDS,

30% (v/v) glycerol, and 2.5% (w/v) iodoacetamide, pH 6.8) for a further 30 min. The IPG strips were placed on 5-20% gradient SDS–PAGE gels and electrophoresed for 1h at 25 mA. After electrophoresis, the gels were stained with Bio-Safe Coomassie Blue (Bio-Rad).

Sub-cellular fractionation and Immunoblot analysis

Sub-cellular fractionation was performed as previously described (Yoshida et al., 2006). Purity of the fractions was analyzed by electrophoresis through SDS–PAGE gels and transferred electrophoretically onto 0.45-μm PVDF membranes (Millipore, Billerica, MD). The blots were probed with anti-FLAG (Sigma), anti-Topo Ⅱα (MBL) and anti-vinculin (Chemicon) for 2 h at room temperature and then with secondary antibodies coupled to horseradish peroxidase (ECL anti- mouse or rabbit IgG; Amersham) for 1 h at room temperature. The blots were developed using the enhanced chemiluminescent reagent SuperSignal (Pierce, Rockford, IL) and exposed to Kodak X-

OMAT film, according to the manufacturer’s instructions.

Figure legends

Figure S1, Two-dimensional SDS-PAGE analysis of GST, GST-NES1 and GST-NES2. (a)

Affinity-purified GST fusion proteins (GST-NES1 and GST-NES2) and GST were subjected to

SDS-PAGE in a 5-20% gradient gel, and stained with Bio-Safe Coomassie Blue staining. (b) As the first dimension, aliquots of highly purified GST-NES1 (upper panel) or GST-NES2 (lower panel) along with GST were subjected to iso-electric focusing in pre-cast immobilized pH gradient gel strips (pH 3-10) and SDS-PAGE as the second dimension was performed in a 5-20% gradient gel.

Protein spots were visualized with Bio-Safe Coomassie Blue staining. Figure S2,

HeLa cells were transfected with expression plasmids for the indicated GFP fusion proteins, and treated with or without LMB. Immunofluorescence microscopy for anti-GFP and Hoechst (blue) was then performed.

Figure S3,

Sub-cellular fractionation of COS-7 cells transiently expressing FLAG-BRCA2 (WT) and FLAG-

BRCA2 (L1384A). Equal numbers of cells were harvested and fractionated into cytoplasmic (C) and nuclear (N) extracts. Equal amounts of C and N extracts, together with total cell lysates (T) were analyzed with SDS-PAGE and probed with anti-FLAG antibody, anti-TopoⅡα antibody as a nuclear control and anti-vinculin antibody as a cytoplasmic control. The C/N ratio of FLAG-

BRCA2 (WT) is >1, however that of mutant-type BRCA2 (L1384A) is < 1, suggesting that the exogenous BRCA2 protein was more abundant in cytoplasmic extracts than in nuclear extracts from the cells with wild-type FLAG-BRCA2, however the exogenous BRCA2 protein was less abundant in cytoplasmic extracts of the cells with mutant-type BRCA2 (L1384A).