Introduction -

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Nematode Strains
The wild type strain used was the C. elegans Bristol strain N2. The genotypes of baf-1
mutant worm strains are: pna-1(t1499) (GE2249) or pna-1(t1639) (GE2576), unc-
32(e189)/qC1 dpy-19(e1259ts), glp-1(q339); him-3(e1147) (Gönczy et al, 1999).
Based on our findings we re-named pna-1 baf-1. Strains XA3556 (unc-119(ed3)
qaEx3556[unc-119(+), pie-1::GFP::baf-1], XA3557 (unc-119(ed3) qaEx3557[unc-
119(+), pie-1::GFP::baf-1(t1639)], BN5 (unc-119(ed3) bqEx5[unc-119(+), pie-
1::GFP::vrk-1], BN8 and BN9 (unc-119(ed3) bqEx8/9[unc-119(+), pie-1::LAP::vrk-
1] were generated by microparticle bombardment of DP38 unc-119(ed3) (Praitis et al,
2001) with plasmids pPAG20-baf-1, pPAG20-baf-1(t1639), pID vrk-1 and pPGLv.1
vrk-1, respectively. Strains XA3558 (baf-1(t1639); GFP::-tubulin), XA3559 (baf-
1(t1639); YFP::LMN-1) and XA3560 (baf-1(t1639); GFP::LEM-2) were obtained by
crossing GE2576 with WH204 (GFP::-tubulin, (Strome et al, 2001)), XA3502
(YFP::LMN-1), and XA3507 (GFP::LEM-2) (Galy et al, 2003), respectively. Other
worm strains used: AZ212 (GFP::hisH2B) (Praitis et al, 2001), XA3501 (GFP::-
tubulin; GFP::hisH2B), XA3504 (GFP::EMR-1) (Askjaer et al, 2002), and XA3506
(GFP::NPP-5, (Franz et al, 2005)). All strains were cultured using standard C.
elegans methods (Stiernagle, 2006).

Plasmid Construction
PCR-amplified wild type baf-1 and baf-1(t1639) genomic DNA sequences were
inserted downstream of the gfp sequence in the pie-1-based germline expression
vector pPAG20 (Askjaer et al, 2002) to generate vectors pPAG20-baf-1 and pPAG20-
baf-1(t1639). PCR-amplified vrk-1 genomic DNA sequence was inserted into pID-
3.01B (Pellettieri et al, 2003) and pPGLv.1 by Gateway cloning (Invitrogen) to
generate pID vrk-1 and pPGLv.1 vrk-1, respectively. Plasmid pPGLv.1 was
constructed by inserting a PCR fragment encoding TEV-S-peptide amplified from
pIC26 (Cheeseman et al, 2004) into the unique SgrAI site of pID-3.01B.
For RNAi constructs the following sequences were obtained by PCR and inserted into
pPD129.36 L4440 vector: Full-length baf-1 genomic DNA, baf-1 genomic DNA

corresponding to 3’UTR nucleotides (nt) 1-336 downstream of the stop codon, and
vrk-1 ORF nt 835-1625. As a negative control, the empty pPD129.36 vector was used.
Plasmids pQE30-VRK-1 and pQE60zz-VRK-1 were made by insertion of RT-PCR-
amplified full-length vrk-1 cDNA into BamHI/HindIII sites of pQE30 and BamHI site
of pQE60zz, respectively. pGEX-VRK-1-His was constructed by insertion of a
BamHI/XbaI fragment from pQE30-VRK-1 and a XbaI/HindIII fragment from
pQE60zz-VRK-1 into BamHI/HindIII sites of pGEX-KG.
Plasmid pQE82l-BAF-1 was generated by insertion of RT-PCR amplified full-length
baf-1 cDNA into BamHI site of pQE82l. Vector zz80N-BAF-1 was made by insertion
of baf-1 cDNA into BamHI site of zz-80N.

Recombinant Proteins and Antibodies
His-VRK-1 was purified under denaturing conditions according to standard
procedures (Qiagen), dialyzed and injected into rabbits. For affinity purification of
anti-VRK-1 antibodies, GST-VRK-1-His purified under native conditions with
Glutathione Sepharose 4 (Amersham Pharmacia) was immobilized on a CNBr-
activated Sepharose 4B column (Amersham Pharmacia) and incubated with anti-
VRK-1 antiserum. Anti-VRK-1 antibodies were eluted with 0.1 M glycine pH 2.5.
For protein kinase assays native GST-VRK-1-His was tandem purified over
glutathione and cobalt (BD Bioscience Clontech) columns before dialysis against
phosphate buffered saline (PBS) containing 8.7 % (v/v) glycerol.
His-BAF-1 was purified under denaturing conditions in the presence of 6 M guanidine
hydrochloride, dialyzed against PBS containing 8.7 % (v/v) glycerol and was used for
immunizing rabbits. zz-BAF-1-His for kinase assays was purified under native
conditions according to standard procedures and dialyzed in PBS containing 8.7 %
(v/v) glycerol. All proteins were expressed in E. coli.

Antibodies were diluted as follows: Anti-VRK-1 antiserum and affinity purified
antibodies, 1:200; anti-BAF-1 antiserum, 1:250; anti-Nup96 antiserum (Galy et al,
2003), 1:500; anti-LEM-2 antiserum 3597 (Lee et al, 2000), 1:100; anti-EMR-1
antiserum 3598 (Lee et al, 2000), 1:100; monoclonal antibody 414 against
nucleoporins (Jackson Immunoresearch Laboratories, West Grove, PA), 1:250-1:400;
Cy5-conjugated donkey anti-rat antibody (Jackson), 1:500; goat anti-mouse Alexa

Fluor 488 and 546 and goat anti-rabbit Alexa Fluor 633 antibodies (Molecular Probes,
Eugene, OR), 1:1000. Hoechst 33258 (Hoechst, Germany) was used at 1-1.25 g/ml
final concentration. Confocal images were obtained with a Leica AOBS SP2

Transmission Electron Microscopy
C. elegans hermaphrodites fed bacteria expressing either control or vrk-1 dsRNA
were cryo-immobilized immediately using a Leica EM PACT high-pressure freezer
(Leica, Vienna, Austria) and processed as described (Franz et al, 2005). Analysis of
baf-1(t1639) and baf-1(RNAi) embryos was performed with the following
modifications. Worms were transferred to planchettes filled with 20% BSA and were
rapidly frozen using an EM PACT2 (Leica, Vienna, Austria). Specimens were freeze
substituted for 48 h at –90oC in acetone containing 2% osmium tetroxide, 0.1% uranyl
acetate and 5% water (Walther and Ziegler, 2002). They were then brought to –30oC
for 3 h and then gradually to room temperature (5oC/h).

Affinity purified anti-VRK-1 antibody (25 µg) or rabbit control IgG (25 µg, Santa
Cruz Biotechnology) was coupled to 50 µl AffiPrep Protein A support (BioRad) using
20 mM dimethylpimelimidate and washed in the following order with 1) 0.2 M
ethanolamine, 0.2 M NaCl, pH 8.5, 2) lysis buffer (50 mM HEPES, 1 mM EGTA, 1
mM MgCl2, 100 mM KCl, 10% glycerol, 0.05% Nonidet P40, pH 7.4), 3) 0.1 M
glycine, pH 2.5, 4) lysis buffer. Approximately 600 µl of embryos obtained by
bleaching N2 adults (Stiernagle, 2006) were resuspended in lysis buffer (without
Nonidet P40) and frozen in N2. Embryos were ground in a pre-chilled mortar in a total
volume   of   1.2   ml   lysis   buffer   containing   protease   inhibitors   (1   mM
phenylmethylsufonyl fluoride, 1 mM benzamidine, Roche Complete Mini EDTA-
free) and 0.05% Nonidet P40 followed by mild sonication on ice until visual
inspection under a dissection stereomicroscope revealed complete breakage of
embryos. Extracts were cleared by centrifugation at 10,000 g for 15 min and 250 µl
supernatant were incubated with each antibody column by rotation for 12 h at 4C.
Unbound material was collected and columns were washed with 80 bed volumes of
lysis buffer before bound proteins were eluted with 100 µl 3.5 M MgCl2, precipitated

with acetone and separated by 15% SDS-PAGE. Proteins were transferred to
Immobilon P membrane (Millipore), which was probed with anti-BAF-1 antiserum
diluted 1:500 in PBS containing 0.05% Tween-20 and 3% low-fat milk.

2-Dimensional Gel Analysis
Worms were grown as specified in Results and bleached to obtain the embryos.
Embryos were frozen in N2 and ground for 10 min, re-suspended in buffer containing
7 M urea, 2 M thio-urea, 4% CHAPS, 5 mM tris-butyl-phosphine (TBP), 0.25% SDS
and 2% biolyte. Proteins were precipitated with acetone and methanol overnight at -
80°C and centrifuged at 6000 g for 30 min. The pellets were washed twice in acetone
and re-suspended by vortexing at 23°C for 4 h in buffer containing 9 M urea, 4%
CHAPS, 100 mM DTT, 1% biolyte, protease inhibitors (Roche Protease Inhibitors
Cocktail, 1:100 v/v) and phosphatase inhibitors (Sigma Phosphatase Inhibitors I and
II, 1:100 v/v). Non-soluble proteins and debris were removed by centrifugation at
4000 g for 30 min. 10 µg total proteins of each sample were applied to 7 cm 3-10 IPG
strips (Linear gradient, GE Healthcare, Little Chalfont, UK), re-hydrated and focused
in a Protean IEF Cell (BioRad, Hercules, CA). For the second dimension, strips were
first equilibrated for 15 min in equilibration buffer (6 M urea, 30% glycerol, 5% SDS,
0.05 M Tris) containing 1% DTT, followed by 15 min in equilibration buffer
containing 4% (w/v) iodoacetamide, then resolved on 15% SDS-PAGE gels. Proteins
were transferred to nitrocellulose membranes that were probed with anti-BAF-1
antiserum diluted 1:1000 in PBS containing 0.1% Tween-20 and 3% low-fat milk.
For  phosphatase treatment wild type frozen embryos were ground and re-suspended
in phosphatase buffer (50 mM Tris (pH 7.5), 50 mM NaCl, 0.1 mM MnCl2, 2 mM
DTT, 0.075% NP-40 and protease inhibitors) and split in two halves. Phosphatase
inhibitors were added to the control sample, while 1000 units  protein phosphatase
(New England Biolabs) and 2 mM MnCl2 (final) was added to the other half. Samples
were incubated with gentle shaking at 30°C for 2 h followed by precipitation and 2-D

In vitro kinase assays
Kinase assays containing 0 or 0.5 µM GST-VRK-1-His and 20 µM of zz-BAF-1-His
were performed at room temperature in 50 µl reactions containing 20 mM Tris (pH

7.5), 5 mM MgCl2, 1 mM DTT, protease and phosphatase inhibitors. Reaction was
initiated with 0.5 µl of 10 µCi/µl -32PATP and stopped after 60 or 120 min by
addition of SDS-PAGE sample buffer. Proteins were resolved by 12% SDS-PAGE
and phosphorylation was detected by autoradiography.


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