Materials and methods Mouse Emi1 was amplified by PCR using a 13 dpc embryo cDNA library and oligonucleotides 5′-AGGAATTCATGAAGTGTTTTAATTGCAACCCT G-3′ (forward) and 5′-GGTCGACTCACAATCTTTGTAAGTTCTTTTTA C-3′ (reverse) and subcloned into the EcoRI and SalI sites of either pCDNA3-myc or pGEX4T1 expression vectors for myc- or GST-tagged Emi1, respectively. Oligonucleotides were derived from the mouse Emi1 homologue deposited in the NCBI database (Fbxo5: NM_025995). Additional oligonucleotides used were as follows: 5-GGTCGACTCATAGGTGCTCCAGGCCCAT-3′ (reverse) for GST-Emi1_1–181, 5′-AGGAATTCATGCAGCGAGTCATTGAAAGC-3′ (forward) for Emi1_236–383, 5′-GGTCGACTCAGGCTTTGAGGCTTTCGTTG-3′ (reverse) for Emi1_236–313. Point mutations in Emi1 were introduced by using mutated oligonucleotides and PCR amplification. Pfu polymerase (Stratagene) was used for all amplifications and constructs sequences were verified by direct sequencing. The vector pMT2-HAp90^Rsk2 was a generous gift of Dr Mortin Frodin.

Expression and purification of GST fusion proteins Plasmids (pGEX-) containing GST fusion proteins were transformed into the Escherichia coli BL21 strain, and grown at 30°C in LB medium to an OD₆₀₀=0.6 before induction with 0.5 mM isopropyl-β-thiogalactopyranoside (IPTG, Sigma-Aldrich) for 3 h. GST fusion proteins were purified from bacterial lysates on glutathione-agarose (Sigma-Aldrich) as previously described (Sette et al, 1998) and analysed by SDS–PAGE and Coomassie blue staining to test purity and integrity.

Cell culture and transfections Hek293 cells were maintained in Dulbecco's medium supplemented with 10% fetal bovine serum (FBS) (Gibco BRL) in 90 mm dishes. Subconfluent monolayers were processed for CaPO₄ transfection with 1–10 μg of the appropriate plasmids or by Fugene (Stratagene) with 0.2–2 μg of the appropriate plasmids as previously described (Sette et al, 2002). At 24–48 h after transfection, cells were harvested in lysis buffer (50 mM Hepes, pH 7.5, 75 mM NaCl, 10 mM β-glycerophosphate, 2 mM EGTA, 15 mM MgCl₂, 0.1 mM sodium orthovanadate, 1 mM DTT, 0.5% Triton X-100, protease inhibitor cocktail (Sigma-Aldrich)) and incubated for 10 min on ice. Lysates were centrifuged for 10 min at 10 000 g at 4°C and used for further analysis. Protein concentration was determined using a protein assay kit (Bio-Rad) following the manufacturer's instructions.

FACS sorting Transfected cells were separated based on size (forward scatter) and green fluorescence (GFP-positive) using a FACSVantage cell sorter (Beckton and Dickinson). Purity of GFP-positive and -negative populations was >98%. Sorted cells were used for Western blot analysis as described below.

Pull-down assays Cell extracts (500 μg of total proteins) were added to 2 μg of GST fusion protein adsorbed on glutathione-agarose (Sigma-Aldrich) in 250 μl (final volume) of lysis buffer supplemented with 0.05% bovine serum albumin (BSA). After incubation for 90 min at 4°C under constant shaking, beads were washed three times with lysis buffer without Triton X-100, and absorbed proteins were eluted in SDS sample buffer (62.5 mM Tris–HCl, pH 6.8, 10% glycerol, 2% (wt/vol) SDS, 0.7 M 2-mercaptoethanol and 0.0025% (wt/vol) bromophenol blue) and resolved on a 10% SDS–PAGE for subsequent Western blot analysis.

Immunoprecipitation assay Cell extracts (500 μg of total proteins) were incubated with 1 μg of anti-myc antibody for 2 h at 4°C under constant shaking. Protein A–Sepharose or protein G–Sepharose (Sigma-Aldrich) was preadsorbed with 0.05% BSA before incubation with the immunocomplexes for an additional hour. Hence, beads were washed three times with lysis buffer and absorbed proteins were eluted in SDS sample buffer for Western blot analysis.

Kinase assays For p90^Rsk2 assays, 1 μg of each GST-Emi1 fusion protein was incubated at 30°C for 20 min with the purified active form of the kinase (5 U, Upstate Biotechnology) in reaction buffer: 50 mM Hepes, pH 7.4, 10 mM MgCl₂, 1 mM EGTA, 1 mM DTT, 10 mM β-glycerophosphate, 0.5 mM NaVO₄, 50 μM ATP and 5 μCi of ³²P-γ-ATP. In some experiments, GST fusion proteins were phosphorylated while still bound to the GSH-agarose beads and at the end of the incubation the kinases were washed by rinsing three times with an excess of kinase buffer (without label) before using the proteins for pull-down assays. H1 kinase assays were performed on cell extracts of GFP-positive Hek293 cells as previously described (Bhatt and Ferrell, 1999).

Western blot analysis Cell extracts or immunoprecipitated proteins were diluted in SDS sample buffer as described above and boiled for 5 min. For oocyte extracts, 300 metaphase II oocytes/sample were collected and immediately frozen in sample buffer. After thawing, oocytes were sonicated and boiled before loading. Proteins were separated on 10% SDS–PAGE gels and transferred to polyvinylidene fluoride Immobilon-P membranes (Millipore) using a semidry blotting apparatus (Bio-Rad). The membranes were saturated with 5% nonfat dry milk in PBS containing 0.1% Tween 20 for 1 h at room temperature, and incubated with the following primary antibodies (1:1000 dilution) overnight at 4°C: mouse anti-HA (for HA-p90^Rsk2, from BabCO Berkeley antibody company); rabbit anti-actin (Sigma-Aldrich); mouse anti-Myc (for myc-Emi1); rabbit anti-Emi1 (Gentaur); goat anti-p90^Rsk2, rabbit anti-Cdc20, rabbit anti-cyclin A2, mouse anti-cyclin-B1. Primary antibodies were all from SantaCruz Biotechnology, unless specified otherwise. Secondary anti-mouse or anti-rabbit IgGs conjugated to horseradish peroxidase (Amersham) were incubated with the membranes for 1 h at room temperature at a 1:10 000 dilution in PBS containing 0.1% Tween 20. Immunostained bands were detected by chemiluminescent method (SantaCruz Biotechnology).

Immunofluorescence analysis Oocytes were processed for immunofluorescence analysis using anti-tubulin antibody (1:100, Sigma-Aldrich) or anti-Emi1 antibody (1:200, Gentaur) or anti-p90^Rsk2 antibody (1:200, SantaCruz Biotechnology) as previously documented (Sette et al, 2002).

 

 

Acknowledgments

CERTIFICATE OF ANALYSIS