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
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
cell debris. The supernatants were loaded onto a Glutathione Sepharose 4B column (MicroSpin
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).