Germany). Brieﬂy, 2 10
cells were nucleofected using the
Amaxa solution kit V, 4 m
M of siRNA smartpool pp60
Lyn (Dharmacon Inc., Lafayette, CO, USA) and the program
X-01 following the Amaxa guidelines. Cells were lysed 48 h
post nucleofection. pcDNA3 containing wild-type NPM-ALK
or the Y338F, Y342F, Y343F, Y418F and Y664F mutants
were already described (Duyster et al., 2001; Cussac et al.,
2004). pcDNA3-EGFP-Rac1T17N was a generous gift from
Dr K Hahn (University of North Carolina, Chapel Hill,
NC, USA). pCI2.F.hVav3.WT, pCI2.F.hVav3.Y173F, pCI2.F.
hVav3.L211Q, pCDNA3.F.hVav3R697A, and pCMS3.H1P
and pCMS3.H1P.shVav3 that contain a separate transcrip-
tional cassette driving Green Fluorescent Protein (GFP)
expression allowing easy identiﬁcation of transfected cells
were already described (Zakaria et al., 2004; Charvet et al.,
Cell lysis, immunoprecipitation and immunoblotting
Total proteins were extracted with lysis buffer (50 m
base pH 8, 150 m
M NaCl, 5 mM EGTA, 1% Nonidet P-40,
M PMSF, 25 mM NaF, 2 mM Na
and 2 mgml
aprotinin). For lysates from NPM-ALK positive
or negative lymph nodes from ALCLs patients, frozen tissues
were sonicated in detergent-free lysis buffer, cleared by
centrifugation and proteins in the supernatant quantitated
with the Bio-Rad protein assay (Bio-Rad, Munich, Germany).
For immunoprecipitations, clariﬁed homogenates were incu-
bated overnight at 4 1C with suited antibodies and a mix of
protein A/G sepharose beads. After washes, proteins were
eluted with Laemmli buffer and analysed by SDS–PAGE
followed by western blotting on Immobilon-P membranes
(Millipore, Billerica, MA, USA). Immunoreactive bands were
detected by chemiluminescence with the SuperSignal detection
system (Pierce Chemical Co, Rockford, IL, USA).
GTPases pull-down assays
The amounts of GTP-bound active Rac1, Cdc42 or RhoA
were determinated by pull-down as previously described
(Benard and Bokoch, 2002). Brieﬂy, cells were lysed in ice-
cold lysis buffer (50 m
M Tris-base pH 7.4, 500 mM NaCl,
, 2.5 mM EGTA, 1% Triton X-100, 0.5% sodium
deoxycholate, 0.1% SDS, 1 m
M PMSF, 50 mM NaF, 1 mM
leupeptin and 2 mgml
clariﬁed lysates were incubated with 30 mg GST-PBD (PAK1
Binding Domain for Rac1 and Cdc42) or GST-RBD
(Rhotekin Binding Domain for RhoA) bound to glutathione
sepharose at 4 1C for 30 min. Beads were washed with 50 m
Tris-base pH 7.4, 150 mM NaCl, 1 mM MgCl
1% triton X-100 and GTP-bound GTPases eluted with
Laemmli buffer and subjected to SDS–PAGE followed by
Cell invasion assay
To assess the role of Rac1 and Vav3 in migration, NIH-3T3
cells were transfected with pCMS3.H1P (expressing GFP),
pCMS3.H1P.shVav3 (expressing GFP and shRNA targeting
Vav3) or pcDNA3.EGFP.Rac1T17N. After 30 h, cells were
seeded in 24-well plates on biocoated Matrigel Invasion
Chambers that consisted in a 8 mm-size pore ﬁlter coated with
a reconstituted basal membrane matrix (Becton Dickinson,
Mountain View, CA, USA). Migration proceeded for 17 h at
37 1C. Then, cells on the upper side of the ﬁlters were scrapped
with a cotton swab and cells positive for GFP were scored. A
correction index was applied to the raw values, it corresponds
to the growth of the various transfectants over 17 h. Results
are expressed as percentages of control cells expressing GFP
We thank Dr Popoff for Clostridium lethal toxins. We are
grateful to Dr H Tronche
re, Dr S Manenti, Dr MP Gratacap,
Dr C Racaud-Sultan and Dr M Plantavid for helpful
discussions. AC and DR were ﬁnanced by the ‘Ministe
la Recherche et de la Technologie’ and the ‘Association pour la
Recherche sur le Cancer’. This work was supported by grants
from the INSERM, ARC, ARECA, La Ligue contre le
Cancer, the ‘Cance
ropoˆ le Grand Sud-Ouest’ and the ‘Institut
National du Cancer’ (INCa), the Re
the ‘Poˆ le de Compe
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A Colomba et al