Functional association of human Ki-1
⁄
57 with pre-mRNA
splicing events
Gustavo C. Bressan
1,2
, Alexandre J. C. Quaresma
1
, Eduardo C. Moraes
1,2
, Adriana O. Manfiolli
3
,
Dario O. Passos
1
, Marcelo D. Gomes
3
and Jo
¨
rg Kobarg
1,2
1 Laborato
´
rio Nacional de Luz Sı
´
ncrotron, Campinas, SP, Brasil
2 Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brasil
3 Departamento de Bioquı
´
mica e Imunologia, Faculdade de Medicina de Ribeira˜o Preto da Universidade de Sa˜ o Paulo, Ribeira˜ o Preto,
Brasil

was able to bind to a poly-U RNA probe in electrophoretic mobility shift
assays. In a classic splicing test, we showed that Ki-1 ⁄ 57 can modify the
splicing site selection of the adenoviral E1A minigene in a dose-dependent
manner. Further confocal and fluorescence microscopy analysis revealed
the localization of enhanced green fluorescent protein–Ki-1 ⁄ 57 to nuclear
bodies involved in RNA processing and or small nuclear ribonucleo-
protein assembly, depending on the cellular methylation status and its
N-terminal region. In summary, our findings suggest that Ki-1 ⁄ 57 is
probably involved in cellular events related to RNA functions, such
as pre-mRNA splicing.
Structured digital abstract
l
MINT-7041074: Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with SF2P32 (uni-
protkb:
Q07021)bytwo hybrid (MI:0018)
l
MINT-7041232: Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with SFRS9 (uni-
protkb:
Q13242)bypull down (MI:0096)
l
MINT-7041203: P80-Coilin (uniprotkb:P38432) and Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) colocalize
(
MI:0403)byfluorescence microscopy (MI:0416)
l
MINT-7041217: SMN (uniprotkb:Q16637) and Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) colocalize
(
MI:0403)byfluorescence microscopy (MI:0416)
l
MINT-7041189: SC-35 (uniprotkb:Q01130) and Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) colocalize
(

and in several nuclear structures [2]. Ki-1 ⁄ 57 was
found to associate with intracellular hyaluronic acid
and other negatively charged molecules in vitro, and
was therefore also named hyaluronic acid-binding
protein 4 [5].
Another human protein, CGI-55, shares 40.7% iden-
tity and 67.4% similarity with Ki-1 ⁄ 57, suggesting that
they could be paralogs and have similar or redundant
functions in human cells. CGI-55 is also a
nucleus ⁄ cytoplasmic shuttling protein [6] and, because
it was described as a protein able to bind to the
3¢-UTR region of the mRNA encoding the type 1 plas-
minogen activator inhibitor, it was also named plas-
minogen activator inhibitor RNA-binding protein 1
[7]. We have recently found that Ki-1 ⁄ 57 and CGI-55
have overlapping interacting protein partners. Among
them are the chromatin remodeling factor chromo-heli-
case DNA-binding domain protein 3 [8], DAXX, and
Topors [6,9]. This suggests that the nuclear functions
of both proteins may be related to transcriptional
activity. Despite the fact that these proteins share
reasonable sequence similarity, Ki-1 ⁄ 57, but not CGI-
55, interacts with the transcription factor MEF2C [10],
p53 [9], and the signaling ⁄ scaffold receptor of activated
protein kinase C (RACK1) [11,12]. Both Ki-1 ⁄ 57 and
CGI-55 mRNAs show ubiquitous expression in all
human tissues tested, and elevated expression in the
heart, muscle, and liver [8]. Ki-1 ⁄ 57 is also expressed
at higher levels in the brain [8].
Both Ki-1 ⁄ 57 and CGI-55 interact with and are

protein hnRNPQ could also be functionally related to
Ki-1 ⁄ 57. hnRNPQ has been reported to be associated
with the regulation of pre-mRNA splicing [18], and
has been previously found to be a novel interacting
l
MINT-7041065: Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with SFRS9 (uni-
protkb:
Q13242)bytwo hybrid (MI:0018)
l
MINT-7041069: Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with YB1 (uni-
protkb:
P67809)bytwo hybrid (MI:0018)
l
MINT-7041079: Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) physically interacts (MI:0915) with HNRPQ
(uniprotkb:
O60506)bytwo hybrid (MI:0018)
l
MINT-7041087: Ki-1 ⁄ 57 (uniprotkb:Q5JVS0) physically interacts (MI:0218) with HNRPQ3
(uniprotkb:
O60506-1), HNRPQ2 (uniprotkb:O60506-2) and HNRPQ-1 (uniprotkb:O60506-3)
by anti bait coimmunoprecipitation (
MI:0006)
G. C. Bressan et al. Human Ki-1 ⁄ 57 and pre-mRNA splicing
FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS 3771
partner and target for Arg methylation by PRMT1
[13,19].
Aiming to confirm the endogenous association of
Ki-1 ⁄ 57 with proteins involved in splicing regulation,
we performed immunoprecipitation assays from HeLa
cell extracts. We confirmed such an association

all interact directly with each other.
Yeast two-hybrid mapping assays
Next, we were interested in knowing the regions of
Ki-1 ⁄ 57 necessary for its interaction with splicing
regulatory proteins. Several N-terminal and C-terminal
Ki-1 ⁄ 57 truncated forms fused to the LexA DNA-
binding domain (Fig. 2F) were cotransformed with
constructs encoding the Ki-1⁄ 57-interacting proteins
fused to a GAL4 activation domain, to test their abil-
ity to interact with each other. Only the full-length
and N-terminal Ki-1 ⁄ 57 constructs were able to inter-
act with the splicing proteins SFRS9, SF2p32, and
YB-1 (Fig. 2G, columns 1–3). This suggests that the
interaction of Ki-1 ⁄ 57 with these molecules may occur
predominantly through its N-terminal region. This
pattern was not verified for hnRNPQ (Fig. 2G, col-
umn 4), as we only observed its interaction with the
full-length Ki-1 ⁄ 57 construct. On the other hand, this
finding may explain why we were not able to identify
hnRNPQ in our yeast two-hybrid screens, where only
the truncated forms of the N-terminus and C-terminus
of Ki-1 ⁄ 57 were used as ‘baits’.
RNA-binding activity of Ki-1
⁄
57 in vitro
Although Ki-1 ⁄ 57 does not have any classic RNA-bind-
ing domains in its amino acid sequence, it has several
Arg ⁄ Gly-rich clusters (RGG-box) (Fig. 3A). The RGG
motif’s importance for the interaction of many
RNA-binding proteins with RNA has already been

to its putative cellular RNA targets may involve
U-rich regions, instead of A-rich regions as reported
for CGI-55 [7].
As we found the N-terminus to be an important
region for the interaction of Ki-1 ⁄ 57 with its protein
A
B
C
E
H G F
D
Fig. 2. Confirmation of the protein–protein interactions among Ki-1 ⁄ 57 and proteins involved in pre-mRNA splicing. (A–C) Immunoprecipita-
tion assays (IP) of endogenous proteins. HeLa cell extracts were immunoprecipitated with G-Sepharose beads and the indicated antibodies.
The obtained protein complexes were analyzed by western blot (WB) as indicated in the figure panels. Arrows indicate the positions of ana-
lyzed proteins. WL, whole cell lysate. Immunoprecipitation with the indicated control antibodies is shown on the right side. (D, E) In vitro
pull-down assays. Recombinant proteins from bacteria [GST, GST–Ki-1 ⁄ 57, GST–hnRNPQ(1–443), 6· His–Ki-1 ⁄ 57] or baculovirus (6·
His–SFRS9) were loaded onto Ni
2+
–nitrilotriacetic acid (6· His-fusion) or glutathione–Sepharose beads (GST-fusion) and incubated with
supernatants of cell lysates as indicated. Arrows indicate the detected proteins. Arrowheads point to the position of the control protein GST.
The additional bands observed correspond to proteolysis degradation products. (F–H) Yeast two-hybrid mapping of Ki-1 ⁄ 57 regions (F) that
interact with the indicated splicing proteins. Black boxes in the diagrams represent the RGG-box motifs present in the sequence of Ki-1 ⁄ 57
(see also Fig. 3A). L40 yeast cells were cotransformed with the plasmids encoding several Ki-1 ⁄ 57 truncated constructs fused to LexA and
the plasmids encoding the prey proteins fused to the GAL4-activating domain (G). Protein–protein interactions were checked through
analysis of reporter gene activation: b-galactosidase activity or capacity to grow on selective minimal medium (in the absence of the amino
acids Trp, Leu, and His) (not shown). (H) Autoactivation control: inability of full-length Ki-1 ⁄ 57 to activate reporter genes in the absence of its
interacting partners.
G. C. Bressan et al. Human Ki-1 ⁄ 57 and pre-mRNA splicing
FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS 3773
partners involved in splicing regulation (Fig. 2G), we

[21]. Depending on the 5¢-splice site selection, the E1A
pre-mRNA may generate five isoforms: 13S, 12S, 11S,
10S, and 9S (Fig. 4A) [22]. These isoforms can be
monitored by RT-PCR followed by agarose gel analy-
sis, where the intensity of each band in the gel directly
correlates with the splicing site selection, which, in
turn, reflects the positive or negative influence of regu-
latory proteins [22,23].
We transiently cotransfected the encoding E1A mini-
gene plasmid with increasing amounts of vectors
expressing the recombinant enhanced green fluorescent
protein (EGFP)–Ki-1 ⁄ 57 in COS7 cells. We observed a
significant effect of EGFP–Ki-1 ⁄ 57 in modifying the
pattern of splicing of E1A mRNA in comparison with
empty pEGFP vector (Fig. 4B). Expression of EGFP–
Ki-1 ⁄ 57 leads to formation of the 10S and 9S iso-
forms, concomitantly with a reduction of 13S isoform
formation, in a dose-dependent way (Fig. 4B, lanes
2–4). This finding strongly suggests the functional
involvement of Ki-1 ⁄ 57 in regulatory mechanisms of
pre-mRNA splicing.
Although we also observed a significant modification
of the E1A mRNA splicing pattern by Ki-1 ⁄ 57(1–222)
and Ki-1 ⁄ 57(122–413), respectively, it only occurred at
the highest plasmid concentrations used (Fig. 4C,D).
Moreover, the effects seemed to be isoform specific for
each Ki-1 ⁄ 57 region, as the formation of 10S mRNA
was only increased by the C-terminal region of
Ki-1 ⁄ 57 although with a lower efficiency in compari-
son with the full length protein (Fig. 4C–E). The influ-

length) or pEGFP–Ki-1 ⁄ 57(1–222) and pEGFP–Ki-1 ⁄ 57(122–413) vectors. The empty pEGFP vector was used to keep constant the DNA con-
centration in each transfection. Splicing activity quantization was performed as described in Experimental procedures. The displayed figures
are representative of at least three independent experiments. Vertical bars in the graphs indicate ± standard deviation. Wherever it exists,
the significance of the difference relative to the control (empty pEGFP vector alone; line 1) is indicated by *P < 0.05. (B–D) Influence of the
overexpression of full-length Ki-1 ⁄ 57 (B) and its N-terminal (C) or C-terminal (D) constructs on E1A splice site selection. Essentially the same
results were obtained in HEK293 cells and when we used a flag-tagged construct of Ki-1 ⁄ 57 (data not shown). (E, F) Treatment ⁄ control band
intensity ratios – comparison of the splicing site selection efficiency of Ki-1 ⁄ 57 and its N-terminal or C-terminal truncated forms. The average
of band intensity values obtained for the isoforms 10S (E) or 9S (F) in (B), (C) and (D) in comparison to the average of the intensities in the
control samples were plotted in the graphs, and represent the fold induction of each isoform in relation to the control. We achieved approxi-
mately 60% transfection efficiency in all experiments performed. Open circles, unspliced pre-mRNA; M, marker.
G. C. Bressan et al. Human Ki-1 ⁄ 57 and pre-mRNA splicing
FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS 3775
Effect of SFRS9 on E1A pre-mRNA splicing in the
presence of Ki-1
⁄
57
SFRS9 and many other Ser ⁄ Arg proteins (SR proteins)
are well known as regulators of E1A pre-mRNA splic-
ing [21,24]. Seeking for a possible functional influence
of Ki-1 ⁄ 57 on SFRS9 splicing activity, we performed
splicing assays in which both proteins were coex-
pressed in COS-7 cells.
When we cotransfected the construct EGFP–SFRS9
alone with the pMTE1A vector, we observed a strong
inhibitory effect on the formation of the 12S and 10S
mRNAs (Fig. 5, lanes 2 and 3), but, similarly to what
was found for EGFP–Ki-1 ⁄ 57, we also observed stimu-
latory activity in generating the 9S isoform (Fig. 5,
lanes 2 and 3). This finding may suggest that although
both proteins may act together in selecting the most

displayed by the endogenous Ki-1 ⁄ 57 in HeLa cells
[13] (data not shown). We then decided to use the
recombinant EGFP-fused form of Ki-1 ⁄ 57 in our con-
focal analysis, mainly because of the insufficient qual-
ity of the images obtained by labeling the endogenous
Ki-1 ⁄ 57 with monoclonal antibodies. We noticed that
Fig. 5. Effect of Ki-1 ⁄ 57 on SFRS9 activity. COS7 cells were transiently cotransfected with an E1A minigene-encoding plasmid [21,23], an
empty EGFP vector, and increasing amounts of pEGFP vectors encoding full-lengths constructs for SFRS9 or Ki-1 ⁄ 57 (1·,4lg; 2·,8lg; 3·,
12 lg). The empty pEGFP vector was used to keep constant the DNA concentration in each transfection. Splicing activity quantization was
performed as described in Experimental procedures. The displayed figures are representative of at least three independent experiments. Ver-
tical bars in the graphs indicate ± standard deviation. Wherever it exists, the significance of the difference relative to the control (empty
pEGFP vector alone; line 1) is indicated by *P < 0.05. Lanes 2 and 3 display the activity of SFRS9 alone, whereas lines 4–6 (darker gray bars)
show the effect of the increasing amounts of Ki-1 ⁄ 57. The white triangle indicates that the value plotted for the 10S isoform in line 6 is dif-
ferent (P < 0.05) to that in line 4. We achieved approximately 60% transfection efficiency in all experiments performed. Open circles, unsp-
liced pre-mRNA; M, marker. The expression of cotransfected Ki-1 ⁄ 57 and SFRS9 was controlled by RT-PCR and is shown in Fig. S1.
Human Ki-1 ⁄ 57 and pre-mRNA splicing G. C. Bressan et al.
3776 FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS
the most evident dot-forming Ki-1 ⁄ 57 in the nuclei of
Adox-treated cells seemed to be related to nucleoli,
mainly because of the well-known large area that this
structure occupies in the cell nucleus.
Although EGFP–Ki-1 ⁄ 57 shows a diffuse distribu-
tion throughout the nucleus, it showed a stronger
signal that colocalizes with the staining of the nucleoli
marker nucleophosmin (Fig. 6Aiii) in Adox-treated
cells. This suggests that the methylation status of
Ki-1 ⁄ 57 is important for its colocalization to this
nuclear subcompartment.
Besides the larger, nucleolar-associated bodies, we
also observed, in the Adox-treated cell nuclei, several

Interestingly, we found, through confocal analyses,
that EGFP–Ki-1 ⁄ 57 was again localized in a diffusive
fashion throughout the nucleoplasm, but showed
stronger spotted staining that colocalized with the
Cajal body protein marker p80-coilin in the nucleus of
HEK293 cells treated with Adox (Fig. 7Ai–iii). This
finding may, in addition, strengthen the hypothesis of
the involvement of Ki-1 ⁄ 57 in pre-mRNA processing
events.
The GEMS are regions enriched with the survival of
motor neurons (SMN) protein complexes and are con-
sidered to be Cajal body-like domains [25]. Although
they may be found as distinct structures, they can also
be found colocalized. This suggests that they are
functionally related [28,29]. However, an interesting
particularity of the SMN protein is its demand for the
presence of Arg ⁄ Gly-rich regions in most of its inter-
Fig. 6. Localization of Ki-1 ⁄ 57 to nucleoli
and splicing speckles. HEK293 cells were
transfected with EGFP–Ki-1 ⁄ 57 and treated
or not treated with the methylation inhibitor
Adox. After fixation, the cells were immuno-
stained with the antibodies against the
nuclear proteins nucleophosmin (NPM;
marker protein of nucleoli) or SC-35 (marker
protein of speckles), and analyzed by laser-
scanning confocal microscopy. (A) Partial
colocalization of EGFP–Ki-1 ⁄ 57 to nucleoli
(nucleophosmin) in Adox-treated cells (Aiii),
but not in the control cells (Ajjj). (B) Partial

cated forms used in the mapping studies described
before (Figs 2G, 3B and 4C–D). Through fluorescence
microscopy analysis of transfected HEK293 cells, we
observed that all tested C-terminal constructs showed
similar nuclear and cytoplasmic localization as
observed for full-length Ki-1 ⁄ 57 (Fig. 8D–F). In turn,
the N-terminal construct displayed an exclusively
nuclear localization that, after careful analysis, could
be found in a few regions consisting of nuclear bodies
(Fig. 8C). This may suggest that the targeting of
Ki-1 ⁄ 57 to nuclear subdomains requires its N-terminal
region. On the other hand, when we treated the
HEK293 cells with Adox, we observed a small but sig-
nificant change in the localization of the C-terminal
construct. In the majority of analyzed cells Ki-1 ⁄
57(122–413) was seen more predominantly in the
nuclear compartment, in contrast to the diffusely
nuclear ⁄ cytoplasmic distribution observed in control
cells (compare panels I and D in Fig. 8). It is interest-
ing to observe that, upon Adox treatment, the N-ter-
minal construct showed pronounced relocalization
from the nucleoplasm to several well-defined nuclear
bodies (Fig. 8H), as observed for the full-length
Ki-1 ⁄ 57 construct (Fig. 8G). More than 90% and 98%
of the cells transfected with full-length EGFP–Ki-1 ⁄ 57
(Figs 6 and 7) and with EGFP–Ki-1 ⁄ 57(1–222) (not
shown), respectively, were found in the nucleus at
nuclear substructures upon Adox treatment (Fig. 8L).
This suggests that the C-terminus of Ki-1 ⁄ 57 is not a
required region for its association with these nuclear

otes, transcription and pre-mRNA maturation events
(5¢-capping, splicing, 3¢-end processing and polyadeny-
lation) occur cotranscriptionally and that the machin-
eries responsible for these activities are functionally
and physically associated [31,32]. Furthermore, it could
be speculated that not only the processing ⁄ maturation,
but also the expression, of some subsets of mRNA
may be regulated by Ki-1 ⁄ 57 in a defined cellular
context. Several of the identified Ki-1 ⁄ 57-interacting
proteins are involved in transcriptional control, such
as CHD3, RACK1, p53 and others p53-associated
proteins [8,9,11], thereby reinforcing a putative tran-
scriptional regulation role for Ki-1 ⁄ 57.
Here, as well as in a previous study, Ki-1 ⁄ 57 has
been observed as dot-like structures in the cell nucleus
[2,13]. Growing evidence points to important roles of
these nuclear subdomains, not only as storage spaces
but also as dynamic structures involved in RNA tran-
scription, processing, and maturation [25,27,33,34]. We
further showed the importance of methylation for its
localization at distinct nuclear ‘spots’, and showed that
in control cells, but not in Adox-treated cells, EGFP–
Ki-1 ⁄ 57 partially localizes to nuclear speckles, whereas
in Adox-treated cells it partially localizes to GEMS,
Cajal bodies, and nucleoli. The nuclear speckles are
known to be storage places for pre-mRNA splicing
complexes and, in turn, Cajal bodies and GEMS are
regions involved in snRNA modification, snRNP bio-
genesis, and trafficking of small nucleolar RNPs (small
nucleolar RNPs) ⁄ snRNPs to nucleoli or speckles,

Ki-1/57(1–222) Ki-1/57(122–413) Ki-1/57(151–263) Ki-1/57(264–413)
Fig. 8. The localization of Ki-1 ⁄ 57 to nuclear
bodies depends on its N-terminal region. (A)
Schematic representations of the truncated
Ki-1 ⁄ 57 constructs fused to EGFP, used for
the localization assays in untreated or Adox-
treated HEK293 cells. (B–F) Untreated cells.
The full-length EGFP–Ki-1 ⁄ 57 and its C-ter-
minal constructs (122–413), (151–263) and
(264–413) show a diffuse nuclear and cyto-
plasmic localization (B, D–F), whereas the
N-terminal construct (1–222) shows an
exclusively nuclear localization (C), at dis-
crete nuclear dots (white arrowheads). (G–L)
Adox-treated cells. The full-length EGFP–Ki-
1 ⁄ 57 and its N-terminal construct (1–222)
predominantly show nuclear localization (G,
H), at several nuclear bodies (white arrow-
heads). A small but significant amount of
nuclear relocalization can be observed for
the C-terminal construct (122–413) (I). No
changes were observed for the smaller C-
terminal constructs (151–263) and (264–413)
(J, K). (L) Proportion of Adox-treated cells
containing nuclear bodies. More than 90%
and 98% of the cells transfected with full-
length EGFP–Ki-1 ⁄ 57 and EGFP–Ki-1 ⁄ 57
(1–222), respectively, were found in the
nucleus at several dots. Approximately 100
cells were analyzed in each of three inde-

their splicing activities.
Apart from the presence of the conserved Arg ⁄
Gly-rich clusters in the sequence of Ki-1 ⁄ 57, no other
amino acid sequence signatures are found through the
common computational predictors available on the
internet (data not shown). We did not find any nuclear
localisation signal or nuclear export signal with signifi-
cant scores on these programs, or any known domains
or motifs. Nonetheless, our deletion studies revealed
an interesting pattern of possible functional regions in
Ki-1 ⁄ 57 (Figs 2G and 3B). The Ki-1⁄ 57 C-terminal
region containing the two major RGG-box clusters
seems to be involved in poly-U RNA binding, whereas
the N-terminal region is mainly related to the interac-
tion with the associated pre-mRNA splicing proteins
SFRS9, SFp32, and YB-1. Analyzing the subcellular
localizations of these truncated Ki-1 ⁄ 57 forms in
HEK293 cells treated with the inhibitor of methylation
Adox, we noticed that only the N-terminal construct
was able to localize to nuclear bodies, similarly to
what was observed with the full-length construct.
Therefore, the localization of the N-terminus to
nuclear bodies in Adox-treated cells may be mediated
via protein–protein interactions.
The fact that we found that the N-terminus of
Ki-1 ⁄ 57 seems to functionally mediate protein–protein
interactions with splicing proteins and that the
RG-box-containing C-terminus seems to mediate inter-
actions with RNA is also very interesting; especially in
light of our results obtained with the protein deletions

(obtained from the pBTM vector) into the pEGFPC vector
(Life Technologies Corporation, Carlsbad, CA, USA). The
pACT2 constructs containing the partial cDNAs for YB-1
and SF2p32 correspond to the ‘bait’ plasmid DNAs iso-
lated from the yeast two-hybrid screening previously
reported [9]. The partial or the total cDNA encoding for
Ki-1 ⁄ 57-interacting proteins were fused to GAL4 (pACT or
pGAD vectors) and applied to mapping assays using sev-
eral truncated forms of Ki-1 ⁄ 57 fused to LexA (pBTM116
vector), as described previously [11,12,42].
Cell culture and treatments, total cell lysates,
immunoprecipitations, and preparation of
cytoplasmic and nuclear fractions
Human L540 and HEK293 cells and monkey COS-7 cells
were cultivated under standard conditions as described
Human Ki-1 ⁄ 57 and pre-mRNA splicing G. C. Bressan et al.
3780 FEBS Journal 276 (2009) 3770–3783 ª 2009 The Authors Journal compilation ª 2009 FEBS
previously [11,41]. Transfection was performed by using the
calcium phosphate method. Treatment with 100 lm Adox
was performed as previously described by De Leeuw et al.
[43]. Total lysates were obtained and immunoprecipitated as
described previously [11], and subcellular fractionation of the
L540 cells was performed as previously described [13].
Pull-down assays, western blots, and antibodies
The in vitro pull-down assays and western blots were
performed as previously described [13,41]. The primary anti-
bodies were: anti-green fluorescent protein (rabbit poly-
clonal; Abcam Inc., Cambridge, MA, USA), anti-hnRNPQ
(mAB; Abcam), anti-c-tubulin (mouse mAB; Invitrogen,
Carlsbad, CA, USA), anti-FEZ1 (rabbit polyclonal) [44],

extraction according to the manufacturer’s protocol. cDNA
synthesis was performed using oligodT primer (GE Health-
care, Waukesha, WI, USA) and the Moloney murine leuke-
mia virus reverse transcriptase (Life Technologies
Corporation). The PCRs were performed with the prim-
ers 5¢-ATTATCTGCCACGGAAGGTGT-3¢ (sense) and
5¢-GGATAGCAGGCGCCATTTTA-3¢ (antisense), as pre-
viously described [21]. After separation of the amplification
products on 3% agarose gels containing ethidium bromide,
the band intensities were calculated using the software
image j ( National
Institute of Mental Health, Bethesda, MD, USA). The
intensities of all isoforms were summed, set as 100%, and
used to normalize the intensity of each band.
Microscopy analyses
For the subcellular localization assays, HEK293 cells were
grown on glass coverslips with the required culture med-
ium. The cells were fixed with NaCl ⁄ P
i
containing 2%
paraformaldehyde, permeabilized with 0.3% Triton X-100,
and blocked with NaCl ⁄ P
i
⁄ 2% BSA. The primary antibod-
ies were incubated at room temperature in NaCl ⁄ P
i
⁄ 2%
BSA, and then with the Alexa594-coupled secondary anti-
body (Life Technologies Corporation). Coverslips were
mounted with Prolong gold antifade medium containing

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