Oregon-R and Canton-S were used as a wild-type control. The following mutant and transgenic flies were used: spn-F1, spn-F2, pUASp HA tagged spn-F , ik21 . Germline expression was performed with nanos-Gal4-VP16  and ubiquitous expression with Act5C-GAL4 (Bloomington). Germline clones for ik2 were generated using the ovoD-FLP technique and FRT40A (2L) . To induce expression of FLP recombinase, flies were mated for 24 hours, and second instar larvae were heat shocked in a 37°C water bath for two hours on three consecutive days.
Constructs and transgenic flies
The entire coding sequence of enhanced green fluorescent protein (EGFP) (Invitrogen) was amplified by PCR using modified primers to create a KpnI restriction site at the 5' end (EGFP-KPN-F-5' GGTACCATGGTGAGCAAGGGCGAGGAGC 3') and an Xba I site at the 3' end (EGFP Xba R – 5' TCTAGACTTGTACAGCTCGTCCATGCCG 3'). The resulting PCR product was digested using Kpn I and XbaI and was cloned into pBlueScript to create the EGFP pBlueScript vector. This vector was later used to clone all of the pUASp GFP-tagged constructs. The full length spn-F coding sequence was amplified by PCR from EST (LD01470) using modified primers to create an Xba I restriction site at the 5' end (5' TCTAGAATGGAGGCATCTGCTGCCAAAATC 3') and an Not I site at the 3' end (5' GCGGCCGCCTGGGTCAGAAGTCACC 3'). To create the N-truncated GFP-tagged form of spn-F, the coding sequence from 1–486 bp was amplified by PCR using modified primers to create an XbaI restriction site at the 5' end (5' TCTAGAATGGAGGCATCTGCTGCCAAAATC 3') and an NotI restriction site at the 3' end (5' GCGGCCGCTCATTGGCATCTGGTTCACT 3'). To create the C-truncated GFP-tagged form of spn-F, the coding sequence from 495–1095 bp was amplified by PCR using modified primers to create an XbaI restriction site at the 5' end (5' TCTAGAACGCAGCACTCCCCCAATCCTCACC 3') and an NotI restriction site at the 3' end (5' GCGGCCGCCTGGGTCAGAAGTCACC 3'). To create a full length GFP-tagged form of Ik2, the entire coding sequence of Ik2 was amplified by PCR from EST (SD10041) using modified primers to create an Xba I restriction site at the 5' end (5' TCTAGAATGTCCTTCCTGCGCGGTTCCGTAAGC 3') and an Not I site at the 3' end (5' GCGGCCGCCTAACTACTTTCCAGACTTCCG 3'). All of the above PCR products were digested using Xba I and NotI and were cloned into EGFP-pBlueScript. The resulting pBlueScript vectors were cut using KpnI and NotI and the inserts cloned into pUASp vectors. To create an mCherry-tagged Spn-F, mCherry in pCS2 was amplified by PCR, using modified primers to create Kpn I restriction sites at both ends (5' CGGGGTACC ATGGTGAGCAAGGGCGAGG 3' and 5' CGGGGTACC CTTGTACAGCTCGTCCATGCCGC 3'). The resulting PCR product was digested with Kpn I and then inserted into a pUASp vector containing Spn-F coding sequence from EST (LD-01470). P-element-mediated germline transformation of this construct was carried out according to standard protocols . Ten independent lines were established from each construct.
Ovaries were dissected in PBS, then fixed for 20 minutes (3.8% formaldehyde in PBS and Heptane) and washed 3 × 10-min in PBST (PBS, 0.3% Triton X-100). The ovaries were incubated for 1 h in PBS, 1% Triton X-100, and blocked for 1 hr in 3% BSA, PBST. After overnight incubation at 4°C with primary antibody in appropriate dilution and PBST washes, the ovaries were incubated with secondary antibody for 1 h, washed, and mounted in 50% glycerol. As a primary antibody we used monoclonal mouse anti-Spn-F (1:10; clones 8C10, 4E6 and 10C5). The secondary antibody, Cy3 goat α-mouse, was used at 1:100 (Jackson Immunoresearch). Egg chambers were imaged on a Zeiss LSM510 laser-scanning confocal microscope.
Egg chamber live imaging
Female flies were mated and fed dry yeast for at least 3 days after eclosion. Ovaries and egg chambers were dissected in a Halocarbon oil 700 (Sigma) drop placed on a 24 × 66 mm cover glass. Confocal images were taken within 40 min after the dissection on an Olympus FV1000 laser-scanning confocal microscope.
Cell culture and transfections
S2 or S2R+ cells were cultured in Schneider's cells Drosophila medium (Biolabs Industries, Israel) containing 10% fetal bovine serum (Biolabs industries, Israel) and penicillin (10,000 u/ml)-streptomycin (10 mg/ml)-amphotericin B (0.025 mg/ml) solution (1:100, Biolabs Industries, Israel). Cells were maintained at 25°C under normal atmosphere. 4*106 cells were transfected with 1 μg pUASp-based expression vectors and the Act 5C-Gal4 driver by using Escort IV (Sigma). The transfected cells were cultured in Schneider's cells Drosophila medium without Bovine serum and antibiotics for 24 h. For western blot analysis the cells were treated in SDS sample buffer. For co-localization assay cells were fixed for 15 min (3.8% formalin in PBS), then washed 3 × 10 min in PBS and mounted in 50% glycerol. Cells were imaged on confocal microscope.
Western blot analysis
Proteins were loaded onto a 10% polyacrylamide gel. Following electrophoresis, proteins were transferred to nitrocellulose membranes (PROTRAN, Schleicher & Schuell) for 1 h at 300 mA. The nitrocellulose membranes were blocked by incubation in TTBS (0.2 M Tris, 1.5 M NaCl, 9 mM Tween 20) containing 5% nonfat dry milk, for 30 min at room temperature followed by 1 h incubation in primary antibody. The membranes were washed in TTBS and incubated for 30 min with either a horseradish peroxidase (HRP)-labelled anti-rabbit or anti-mouse antibodies (Amersham) at a 1:2000 dilution each. The signals were visualized using ECL detection kit (biological industries). Primary antibodies used were: anti-HA (1:500, Santa Cruz Biotechnology), polyclonal anti Spn-F (1:1000), anti-GFP (1:1000, Roche diagnostic), anti-Myc (1:1000, Santa Cruz Biotechnology), anti α-tubulin (1:1000, Sigma). For phosphatase treatment, cell extracts were incubated with calf intestinal phosphatase (10 units, New England Biolabs) at 37°C for 15 min. The reaction was stopped by the addition of sample buffer.
Ovaries from GFP-Ik2; HA-Spn-F/nanos- Gal4 or HA-Spn-F/nanos Gal4 expressing flies were dissected in PBS. Lysates were prepared by grinding 10 ovaries for 15 sec in 50 μl cold lysis; PB, 1% NP-40 and protease inhibitors (Sigma). The lysate was spun for 5 min in a microcentrifuge at maximum speed, and 5 μl of the supernatant was set aside for use as a whole cell lysate control. The remaining lysate was pre-cleared with protein A coated beads (Adar Biotech). After a 30 min incubation on ice, the pre-cleared lysates were incubated with anti-GFP (1:250, Roch Diagnostic) and protein A coated beads. After incubation, beads were washed in lysis buffer, resuspended in an equal volume of protein gel loading buffer, and loaded onto 10% polyacrylamide gel. To detect interaction between proteins, western blot with anti-HA antibody was performed.
Cells expressing constructs as described in the text were treated with lysis buffer (PBS, 1%Triton X-100 and protease inhibitors). Pre-cleared extracts were incubated overnight at 4°C with anti-myc (1:250, Santa Cruz) or anti-GFP (1:250, Roche Diagnostic) mouse monoclonal antibodies. Immunocomplexes were recovered by incubation with protein A coated beads (Adar Biotech) for 2 h at 4°C. To detect interaction between proteins, western blot with α-HA antibody or α-myc antibody was performed. As a negative control, the ovarian lysate was precipitated by protein A-coated beads without antibody.
Immunocomplex Kinase Assay
Cells were treated with lysis buffer containing 20 mM Tris-HCl pH 7.5, 12 mM β-glycerophosphate, 150 mM NaCl, 5 mM EGTA, 10 mM NaF, 1% Triton X-100, 1 mM DTT, 1 mM sodium orthovanadate, 1 mM PMSF, and 1.5% aprotinin. The cell extracts were clarified by centrifugation, and the supernatants were immunoprecipitated with anti-GFP. The immunocomplexes were incubated with [γ-32P] ATP and purified His-tagged Spn-F recombinant protein  that was used as a substrate. The kinase assay was done in a final volume of 20 μl of a solution containing 20 mM Tris-HCl pH 7.5, 20 mM MgCl2, and 100 μM ATP. The kinase reactions were stopped by adding SDS sample buffer and analyzed by SDS-PAGE. The gel was exposed to X-ray film for 24 h and visualized by a Fuji LAS3000 image analyzer.
Protein stability assay in S2R+
For studying the effect of increasing levels of Ik2 on Spn-F stability, S2R+ cells were transfected with different concentrations of pUASp-GFP-Ik2 vector (0, 200, 500, 1000 ng) and pUASp-GFP-Spn-F (1000 ng). The expressions of the proteins were driven by Actin-GAL4 (1000 ng). Empty pUASp vector was added to adjust the total amount of expression plasmid to 3000 ng/well. Cell extracts were subjected to western blot analysis with anti-GFP antibody.
Protein Expression and Purification
The open reading frame encoding Spn-F from EST (LD01470 was amplified by PCR using modified primers to create an EcoRI restriction site at the 5' end (5' CGAATTCATGGAGGCATCTGCCTG 3') and HindIII restriction site at the 3' end (5' CGTCAAGCTTTCAGAAGTCACCCAC 3'). The open reading frame encoding Ik2 from EST (SD10041) was amplified by PCR using modified primers to create a BamHI restriction site at the 5' end (5' CGGATCCCATGTCCTTCCTGCGCGG 3') and an SalI restriction site at the 3' end(5' GCCGTCGACCTAACTACTTTCCAGACTTC 3'). The PCR products were digested with the appropriate restriction enzymes and cloned into pMBPHis-Parallel1  vector, containing N terminal MBP-His6 tags followed by a TEV proteolysis site. The plasmids coding for the fusion proteins were overexpressed in BL21 DE3 Escherichia coli cell, grown in 2.4l 2xYT media (containing 200 mg ampicillin). The culture was grown at 37°C to an approximate A600 O.D. of 0.4–0.6. Protein expression was induced by addition of 0.4 mM IPTG (Bio-Labs, USA), and the culture was further incubated for 2 h at 37°C. Cells were harvested at 4°C by centrifugation at 12,000 rpm. The cell pellets were resuspended in lysis buffer (Tris-HCl 20 mM pH 8, NaCl 200 mM, EDTA 1 mM, β-mercaptoethanol 5 MM, sudium azide 0.02%) containing protease inhibitors PMSF (1 mM) and E64 (10 μM) (Sigma, USA). Cells were lysed by sonication (Sonics & Materials, USA). The cell lysates were centrifuged at 12,000 rpm for 25 min. at 4°C. The supernatant solution was loaded onto an Amylose High Flow (New England bio-labs, England) column pre-equilibrated with loading buffer (Tris-HCl 20 mM pH 8, NaCl 200 mM, EDTA 1 mM, β-mercaptoethanol 5 mM,. The protein was eluted with the same buffer with the addition of 10 mM maltose. The fusion protein Ik2-MBPHis was dialysed against loading buffer containing NaCl 50 mM and eluted from an ion exchange HiTrap ANX FF column (Amersham, SwedenS) in a salt gradient of 0.05–1 M NaCl. Ik2-MBPHis was obtained in the unbound fraction. Spn-F fusion protein was further cleaved with TEV (tobacco etch virus) protease in cleavage buffer (Tris-HCl 0.5 M pH 8, EDTA 5 mM, DDT 1 mM) at 4°C overnight. The cleaved Spn-F was dialysed against loading buffer (Tris-HCl 20 mM pH 8, NaCl 200 mM, β-mercaptoethanol 5 mM, sodium azide 0.002%), and loaded onto a nickel HiTrap IMAC FF column (GEHealthcare, USA). The unbound Spn-F was dialysed against loading buffer containing NaCl 50 mM and eluted from an ion exchange HiTrap ANX FF column (Amersham, Sweden) in a salt gradient of 0.05–1 M NaCl. To verify the complete removal of MBPHis tag from Spn-F sample, western analysis with an anti-His antibody was performed; no signal was detected. Furthermore, MALDI-TOF analysis confirmed that the sample contained only pure Spn-F. Finally, the proteins Spn-F and Ik2-MBPHis were dialysed against buffer PBS.
Binding affinities of Spn-F toward Ik2 were measured using the ProteOn XPR36 Protein Interaction Array system (Bio-Rad, USA) based on surface plasmon resonance (SPR) technology [18, 26]. As negative control we used Sec13-MBP protein (Dictybase ID-DDB0235182). A solution of 0.005% Tween 20 in PBS, pH 7.4, was used as running buffer at a flow rate of 30 μl/min, at 25°C. Three channels (GLC sensor chip) were activated for 5 min with a mixture of 0.2 M EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodimide hydrochloride) and 0.05 M sulfo-NHS (solfo-N-hydroxysuccinimide). Immediately after activation of the surfaces, Ik2-MBPHis (1 μg in 10 mM sodium acetate, pH 5.5) and Sec13-MBP (1 μg in 10 mM sodium acetate, pH 4.5) were injected across channel 2 and 3, respectively. This resulted in Ik2-MBPHis coupled at response levels of 1400 RU and Sec13-MBP coupled at response levels of 5500 RU. Channel 1 served as reference. Finally, all 3 channels were blocked with 1 M ethanolamine-HCl (pH 8.5). Spn-F was then injected perpendicular to ligands, at six different concentrations, using a twofold dilution series within a range of 0.1625 to 5 μM. The six concentrations were injected simultaneously at a flow rate of 65 ul/min for 2.5 min of association phase followed by 10 min of dissociation phase at 25°C. The GLC sensor chip was regenerated with short injection of 50 mM NaOH between consecutive measurements. Results are expressed in arbitrary resonance units (RU) with subtraction of RU values from channel 1. Data were analyzed with ProteOn managerTM software, using the Langmuir model (A + B ↔ AB) for fitting kinetic data .