Figure 7. Binding of different MAGE proteins to NSE4a and NSE4b/EID3 proteins. (A) Cell-free extracts of mouse testes were immunoprecipitated with either IgG or anti-NSE4b followed by Western blotting with the indicated antibodies. (B) S-tagged MAGEA1 or MAGEG1 were co-expressed with FLAG-tagged NSE4b in HEK293 cells. In lanes 1–6 and 13–18, extracts were precipitated with S-protein, whereas in lanes 7–12 they were immunoprecipitated with anti-FLAG antibody. (C–H) S-tagged Class I MAGE protein A1 (C), and class II MAGE proteins D4b (D), F1 (E), G1 (F), necdin (G) or vector alone (H) were co- transfected with FLAG-tagged NSE4a (N4a) (lanes 1–3) or NSE4b/EID3 (N4b) (lanes 4–6). Extracts were immunoprecipitated with S-protein and Western blotted with S-HRP and anti-FLAG antibody. doi:10.1371/journal.pone.0017270.g007 for PCR amplification are listed in Supplementary Table S3. To get pGBKT7-MAGEG1(aa55-292) construct NcoI-XhoI fragment of pTriEx4-MAGEG1(aa55-290) clone was inserted into the pGBKT7 vector digested with NcoI-SalI restriction enzymes. EcoRI-XhoI fragment of pTriEx4-NSE4b(aa1-333) was cloned into pGADT7 yeast-2-hybrid vector. The EID2 ORF was amplified with CTC GAG ATG GCA GAC AGC AGT GTC and TCT AGA CTA TTC TCT ATT GAT AAA C primers and inserted into pGEM-T-easy vector (Promega). The pGEM-EID2 Interactions between MAGE and EID Family Proteins construct was cut with XhoI-XbaI restriction enzymes and subcloned into pCI-neo-FLAG vector. Similarly EID2b ORF was cloned into pGEM-T-easy vector (CTC GAG ATG GCG GAG CCG ACT GGG and ACG CGT TCA GTC GGC CAG AGG AC) and then subcloned into pCI-neo-FLAG vector (using XhoI-MluI restriction enzymes). The other constructs were described previously [8,13,18]. Protein-S pull down assays His-S tag-fusion protein extracts from E.coli strain C41 were preincubated with protein S-agarose beads (Merck). Then in vitro- expressed proteins in a total volume of 200 microliter of HEPES buffer were added and incubated overnight [13]. Input, unbound, and bound fractions were separated by SDS-PAGE, transferred to nitrocellulose membranes, and analyzed by phosphorimaging and immunoblotting with anti-His antibody (Sigma). Nse3 structure modeling The crystal structures of MAGE A4 and G1 (PDB entries 2WA0 and 3NW0) were used as the input structures for Nse3 (aa 90 to 310). The I-TASSER server [31] was used to model the Nse3 structure. Site-directed mutagenesis The QuikChange II XL system (Stratagene) was used to create point mutations in the pGBKT7-Nse3(aa1-328), pGBKT7- MAGEG1(aa55-292) and pTriEx4-MAGEG1(aa1-304) plasmids; the primers are listed in Supplementary Tables S1 and S2. Yeast hybrid assays The Gal4-based two-hybrid system was used to analyze Nse3 mutants. Each pGBKT7-Nse3(aa1-328) mutant was cotrans- formed either with pOAD-Nse1(aa1-223) or pACT2-Nse4(aa1- 300) construct. Similarly, pGBKT7-MAGEG1(aa55-292) mutants were cotransformed either with pOAD-hNSE1(aa1-266) or pGADT7-hNSE4b(aa1-333) plasmid. Colonies were inoculated into YPD media and cultivated overnight. 10- and 100-fold dilutions were dropped onto the SD-Leu,-Trp (control) and SD- Leu, -Trp, -His (with 0, 1, 2, 5, 10, 15, 20, 30, 60, 120 mM 3- aminotriazole) plates. Each mutant was cotransformed at least twice into S. cerevisiae MaV203 yeast strain and at least two independent drop tests were carried out from each transformation. In addition, the results and mutant expression levels were verified in another S. cerevisiae Y190 yeast two-hybrid strain. For yeast-3- hybrid tests, three plasmids pGBKT7-Nse3(aa1-328), pACT2- Nse4(aa1-300) and pPM587-Nse1(aa1-232) were cotransformed into PJ69-4a yeast strain and selected on SD-Leu, -Trp, -Ura plates. Drop tests were carried out on SD-Leu, -Trp, -Ura, -His (with 0, 1, 2, 5, 10, 15, 20, 30, 60, 120 mM 3-aminotriazole) plates at 30uC. Generation of Nse3 mutant strains of S. pombe The Nse3 mutant strains were created using Cre recombinase- mediated cassette exchange, as detailed in Watson et al. [32]. The S. pombe strain ‘501’ (ura4-D18, leu1-32, ade6-704, h2) was used to construct the Nse3 base strain with the loxP site integrated 198 bp upstream and the ura4+ marker and loxM3 site integrated 98 bp downstream of the nse3 ORF. A fragment comprising the nse3 ORF, as well as the 198 bp upstream and 98 bp downstream sequences were amplified and cloned into SpeI and SphI sites of pAW6. Site-directed mutagenesis was carried out using Quik- Change Kit (Stratagene). Mutated sequences flanked by loxP and loxM3 sites were then cloned into pAW7 (LEU2+ marker) and PLoS ONE | www.plosone.org 11 February 2011 | Volume 6 | Issue 2 | e17270