Interactions between MAGE and EID Family Proteins Figure 8. Interactions of MAGE proteins with EID1, 2 and 2b. (A) Alignments of the five members of the human EID family. Hatched and grey boxes indicate kleisin motifs and regions of homology between EID1 and 2, respectively. (B–G) S-tagged Class I MAGE protein A1 (B), and class II MAGE proteins D4b (C), F1 (D), G1 (E), necdin (F) or vector alone (G) were co-transfected with FLAG-tagged EID1 (E1) (lanes 1–3), EID2 (E2) (lanes 4–6) or EID2b (E2b) (lanes 7–9) into HEK293. Extracts were immunoprecipitated with S-protein and Western blotted with S-HRP and anti-FLAG antibody. doi:10.1371/journal.pone.0017270.g008 transformed into the Nse3 base strain. ura+, leu+ transformants were selected in the presence of thiamine (i.e. in absence of Cre recombinase expression), grown in nonselective medium for 24 hours, and then plated onto medium containing 5-fluoroorotic acid to select clones in which cassette exchange took place. 5- FOAR and leu2 colonies were picked and the presence of the respective mutations verified by sequencing. Spot tests for sensitivity to DNA damaging agents S. pombe cultures were grown to mid log phase, concentrated to 36107 cells/ml, and serial 6-fold dilutions were spotted onto rich media with or without the indicated dose of DNA-damaging agents. Subsequently, plates were incubated at the indicated temperature for 3–4 days. Mammalian cell culture and luciferase assays HEK293 cells (DSMZ, Germany) were grown in DMEM supplemented with 10% foetal bovine serum and 100 mg/ml penicillin/streptomycin (Invitrogen). Plasmid transfections were carried out using calcium phosphate precipitation. For luciferase assays, cells transfected with pUAS-tk-luc [33] and pHRL-CMV (Promega) and with or without combinations of pSG4-Gal4-mSF- 1-N1 [20], EID and MAGE constructs were processed and luciferase activity determined using the dual luciferase assay kit according to the manufacturer’s instructions (Promega). Antibodies Full-length hNSE4b was expressed in bacteria as a glutathione S-transferase fusion, purified on glutathione Sepharose (GE Healthcare) according to the manufacturer’s instructions, and used to inoculate two rabbits for antibody production (Euro- gentec). Antibodies were affinity purified using antigen immobi- lized with Aminolink Plus coupling gel (Pierce). hNSE1, hNSE2 and hSMC6 antibodies have been described previously [18]. Anti- FLAG M2 (Sigma) and S-HRP (Merck) commercial antibodies were also used in this study. Immunoprecipitations Lysates were made from transfected HEK293 cells by scraping in lysis buffer (50 mM Tris-HCl pH7.5, 0.5% NP40, 40 mM NaCl, 2 mM MgCl2, 16 protease inhibitor cocktail [Roche], 1 mM NEM, 25 U/ml benzonase [Merck]). Lysates were incubated for 30 minutes on ice, cleared by centrifugation at 13000 rpm for 10 minutes and the NaCl concentration adjusted to 150 mM. Agarose beads conjugated to S protein (Merck) or anti- FLAG antibody (Sigma) were mixed with lysates for 4 hours at 4uC. Beads were washed 3 times with wash buffer (50 mM Tris- HCl pH7.5, 150 mM NaCl, protease inhibitors) before beads were resuspended in SDS loading buffer. Lysates for testes were prepared by addition of lysis buffer (50 mM Tris pH7.5, 1% triton, 40 mM NaCl, 2 mM MgCl2, 26 protease inhibitor cocktail [Roche], 1 mM NEM, 25 U/ml benzonase) and 20 strokes with a loose Dounce homogenizer, followed by treatment as above. Lysates were depleted of non- specific binding proteins by incubation with beads cross-linked to rabbit IgG for 1 hour, followed by incubation with the desired antibody for 2 h at 4uC. They were then centrifuged at 15000 rpm for 10 minutes and the supernatant added to protein G beads. Following mixing for 1 h at 4uC, samples were washed and processed as above. For immunoprecipitations performed for mass spectrometry, lysates were prepared as above, but were then incubated with antibodies (anti-SMC6 or IgG) cross-linked to protein A-agarose beads [18]. Following immunoprecipitation, samples were washed as previously, followed by 3 washes in low Tris buffer (4 mM Tris- HCl pH 7.5, 150 mM NaCl, protease inhibitors). Elution was performed by incubation with 200 mM glycine pH 2.5 for 5 min at room temperature. This sample was then neutralised by adding PLoS ONE | www.plosone.org 12 February 2011 | Volume 6 | Issue 2 | e17270