Hydra vulgaris was cultured at a temperature of 18°C in medium containing 0.1 mM KCl, 1 mM NaCl, 0.1 mM MgSO4, 1 mM Tris, and 1 mM CaCl2. The animals were fed regularly with freshly hatched Artemia nauplii.
Antibodies and reagents
Anti-14-3-3 antibody K19 was purchased from Santa Cruz Biotechnology (Santa Cruz) and used at 1:500 to 1:1000. Anti-Tubulin antibody (WA 3, used at 1:10) was a kind gift from Prof. Manfred Schliwa, Munich. Rhodamine-phalloidin was a kind gift from Dr. Ralph Gräf, Munich. Anti-mouse-FITC secondary antibody (used at 1:50) was from Sigma-Aldrich (Hamburg), anti-rabbit-Cy3 secondary antibody (used at 1:500) was from Dianova (Hamburg). Anti-digoxigenin-HRP antibody was purchased from Roche Diagnostics (Mannheim, used 1:1000) and anti-Xpress from Invitrogen (used 1:5000).
Preparation of hydra lysate
Prior to lysis the animals were starved for two days. On the day of lysis they were washed twice in hydra medium, which was then replaced by 400 ml lysis buffer (1% Triton X-100, 1% CHAPS, 2 mM Mg-ATP, 10 μg/ml antipain/leupeptin/pepstatin A/aprotinin, 1 mM pefabloc, 1 mM vanadate, phosphatase inhibitor cocktail (Roche)). Animals were lysed by passing them through a 17 gauge needle and subsequent freezing at -80°C. The lysate was then clarified by centrifugation for 30 min at 30,000 g and 4°C and the supernatant was mixed with 8 mg Bmh1/Bmh2-CH-sepharose 4B for 1 h at 4°C.
14-3-3 affinity column
The 14-3-3 affinity column was prepared essentially as described in Moorhead et al . Briefly, 8 mg recombinant 14-3-3 from Saccharomyces cerevisiae (Bmh1 and Bmh2, 6× His tagged, expressed in E. coli DH5α, purified as in [19, 20]), was incubated with activated CH sepharose at room temperature. Non-reacted, active groups were blocked with 0.1 M Tris/Cl pH 8. A lysate prepared from 100 000 hydra was incubated with 14-3-3 sepharose for 1 h at 4°C. The mixture was then packed into a disposable plastic column (Biorad) and washed with 50 mM HEPES, pH 7.5/0.5 M NaCl/1 mM DTT. To test whether proteins were eluted unspecifically, the column was washed with an unrelated phospho-peptide (-WFYpSFLE-). The 14-3-3 binding proteins were then eluted from the column with 1 mM peptide C (-ARAApSAPA-). The presence of 14-3-3 binding proteins in every fraction was tested in a Far Western Overlay with DIG labelled 14-3-3 proteins.
Mass Spectrometry and EST analysis
20 μg of eluted 14-3-3 binding proteins were separated in an SDS-gel and stained with colloidal coomassie. Single protein bands were cut out of the gel and washed twice with water and twice with 40 mM ammoniumbicarbonate. After two-fold treatment with 50% acetonitrile for 5 min, 10 μg/ml trypsin (Promega) was added and proteins were digested overnight in 40 mM ammoniumbicarbonate at 30°C while shaking. For protein identification probes were directly used for nano-ESI-LC-MS/MS. Each sample was first separated on a C18 reversed phase column via an acetonitrile gradient (Famos-Switchos-Ultimate System and column (75 μm i.d. × 15 cm, packed with C18 PepMap™, 3 μm, 100 Å) by LC Packings) before spectra were recorded on a QSTAR XL mass spectrometer (Applied Biosystems). The resulting spectra where then analysed via the Mascot™ Software (Matrix Science) using the Hydra Protein database (see below).
Peptide prediction from Hydra EST sequences
Hydra EST sequences were downloaded from the EMBL sequence database  and were assembled on the Sputnik comparative genomics platform . These sequences are derived from several cDNA libraries made from whole budding hydra (Hydra EST database). To increase the relative quality of the sequences and to reduce sequence redundancy sequence clustering was performed using the HarvESTer application.
All unigene sequences were compared against known or predicted peptides using the BLASTX algorithm against a non-redundant (Nonred) protein sequence database. Best scoring BLASTX matches exceeding the arbitrary expectation value of 10e-10 were selected and the Hydra coding sequence was extracted from the BLAST output. 1,853 high scoring sequence blocks were selected. These sequence blocks were scored for the relative occurrence of all in-frame hexanucleotide sequences. The concomitant di-codon probability tables were used with the frame finder application to select for the most parsimonious open reading frame from each unigene sequence. This yielded robust peptide sequences even in the absence of a BLASTX homologue.
DIG labelling of 14-3-3 proteins
Recombinant 14-3-3 from yeast (Bmh1 and Bmh2, see above) was labelled with digoxigenin-3-O-methylcarbonyl-e-aminocaproic acid-N-hydroxysuccinimide ester and separated from excess reagent using the digoxygenin protein labeling kit (Roche Diagnostics) according to the manufacturer's protocol. Labelled 14-3-3 protein was diluted to a final concentration of 1 μg/ml in 2 mg/ml BSA and 0.05% sodium azide and stored at 4°C.
Far Western Overlay
The overlay assays were carried out as described by Moorhead et al. . In brief, after SDS-PAGE and Western blotting blots were probed with DIG labelled 14-3-3 proteins and HRP-labelled anti-DIG antibody (Roche Diagnostics) according to the manufacturer's instructions. ECL was used for detection.
For immunoprecipitation experiments, hydra cellular lysates were made in lysis buffer (1% Triton X-100, 1% Chaps, phosphatase- and protease inhibitors). Lysates were incubated with 5 μg of the anti-14-3-3 antibody K19 (Santa Cruz) for 1 h at 4°C. Protein A (Amersham Biosciences) was added and the samples were incubated for an additional hour at 4°C. Samples were then spun down and pellets were washed three times with lysis buffer and one time with Tris buffer. After addition of SDS loading buffer, the proteins were separated by SDS PAGE and subjected to Western blot analysis. In cases when peptide C was used, hydra lysates were incubated with 1 mM peptide C for 2 h at 4°C prior to immunoprecipitation. In samples treated with phosphatase, the lysates were incubated with 200 U λ-phosphatase (New England Biolabs) for 30 min at 30°C prior to immunoprecipitation.
Expression of GFP fusion proteins in Hydra
To transiently express GFP-fusion proteins in hydra, the corresponding genes were introduced into hydra using the PDS-1000/He Particle Delivery System (Biorad) as described in . Briefly, 20 μg of DNA was added to 3 mg of gold particles (1 μm diameter) and precipitated with 0.3 M sodium acetate and 2.5 vol ethanol. Coated particles were washed with 70% ethanol, resuspended in 200 μl ethanol, and spread onto carrier disks according to the manufacturer's instructions. Hydra were collected in petri dishes and as much medium as possible was withdrawn. The animals were then shot 3 times with the gold particles. 2–3 days after transformation animals were screened for expression of the GFP fusion protein.
For immunofluorescence animals were relaxed in 2% urethane for 2 min and then fixed for 1 h at room temperature with Lavdovsky (formaldehyde : acetic acid : ethanol : water 5:2:25:20). After fixation animals were permeabilised in 0.5% Triton X-100 and unspecific binding sites were blocked with 1% BSA/0.1% Triton X-100. Incubation with primary antibody was carried out over night at 4°C in blocking solution, followed by washes with phosphate buffered saline and incubation with fluorescently labelled secondary antibody for 2 h at room temperature. Animals were counterstained with TO-PRO3 (Molecular Probes) and mounted in Vectashield (Vector Laboratories) to prevent bleaching. Animals were analysed by confocal microscopy.
Light optical serial sections were acquired with a Leica (Leica Microsystems, Heidelberg) TCS SP confocal laser scanning microscope equipped with an oil immersion Plan-Apochromat 100/1.4 NA objective lens. Fluorochromes were visualised with an argon laser with excitation wavelengths of 488 nm, emission filter 520–540 nm (for FITC) and with a helium-neon laser with excitation wavelength of 633 nm, and emission filter 660–760 nm (for TO-PRO3 and TRITC). Two fluorochromes and the phase contrast image (transmission filter) were scanned sequentially. Image resolution was 512 × 512 pixel with a pixel size ranging from 195 to 49 nm depending on the selected zoom factor. The axial distance between optical sections was 300–500 nm for zoom factor 4 and 1 μm for zoom factor 1. To obtain an improved signal-to-noise ratio each section image was averaged from four successive scans. The 8-bit greyscale single channel images were overlayed to an RGB image assigning a false colour to each channel, and then assembled into tables using ImageJ 1.32j and Adobe PhotoShop 5.5 software.
14-3-3HyA and 14-3-3HyB were cloned into the plasmid pRSET, expressed in bacteria and purified as described previously . Hybcl-2-like1 was cloned into the vector pGEX and expression of GST-Hybcl-2-like1 or GST-Hyinnexin 1 as a control was induced. Equal amounts of bacterial lysates were incubated with glutathione-sepharose beads for 2 h at room temperature under constant agitation. The beads were sedimented by centrifugation, washed and subsequently incubated with the purified Hydra 14-3-3 proteins for 2 h at room temperature under constant agitation. After centrifugation and washing with PBS, bound proteins were eluted with reduced glutathione and subjected to SDS-PAGE and immunoblotting with anti-Xpress (for proteins expressed from pRSET) and anti-GST antibodies (27457701, GE Healthcare).