dures involving animals were performed in conformity with the animal protocol approved by the Lab Animal Council, National Yang-Ming University. cDNA constructs cDNAs encoding various 14-3-3 isoforms were isolated from a rat brain cDNA library (OriGene) and subcloned into a modified pcDNA3.1 vector (Invitrogen) with a myc tag. The rEag1 cDNA was kindly provided by Dr. Olaf Pongs (Institute fur Neurale, Signalverarbeitung, Zentrum fur Molekulare Neurobiologie, Germany). pSCM138 (difopein/pEYFP-C1, the 14-3-3-binding antagonist) and pSCM174 (the inactive mutant control of pSCM138.) are generous gifts from Dr. Haian Fu (Department of Pharmacology, Emory University School of Medicine, USA). All cDNAs as well as subcloned constructs were verified by DNA sequencing (Genome Research Center, National Yang-Ming University, Taiwan). Yeast two-hybrid screening The DupLEX-A yeast two-hybrid system (OriGene) was used to screen the rat brain cDNA library. The N-terminus of rEag1 (amino acids 1–207) was amplified by PCR and fused in-frame to the coding sequence for the DNA binding protein LexA in the yeast expression plasmid pGilda, which in turn was used as the bait to screen the library. The yeast strain EGY48, which contains the reporter gene LEU2 downstream of the LexA-operator, was sequentially transformed with i) the bait plasmid pGilda-N207, ii) 14-3-3h Interacts with rEag1 the reporter plasmid pSH18-34 (containing the LexA operator- lacZ fusion gene), and iii) an activation domain-fused rat brain cDNA library in pJG4-5 (OriGene) by using the lithium acetate method. After incubating at 30uC for 2–7 days, transformed yeast colonies growing on leucine dropout plates were scored positive for interacting proteins. Positive colonies were further selected by the b-galactosidase assay. Plasmid DNA was extracted from yeast colonies and used to transform the Escherichia coli strain DH5a. Candidate cDNA clones were screened by PCR with pJG4-5- specific primers, followed by online (BLAST) and in-house sequence analyses. Glutathione S-transferase (GST) pull-down assays GST fusion proteins were produced and purified by following the manufacturer’s instruction (Stratagene). In brief, the cDNA fragments encoding the rEag1 amino (N)- or carboxyl (C)-terminus were subcloned into the Escherichia coli expressing pGEX vector and expressed in the Escherichia coli strain BL21. Bacterial cultures were grown at 30uC, induced with 0.1 mM isopropyl-b-D- thiogalactopyranoside (IPTG), and then harvested by centrifuga- tion at 8,0006g for 10 min at 4uC. Cell pellets were resuspended in the B-PER reagent (Pierce) containing 1 mM phenylmethylsul- fonyl fluoride (PMSF) and protease inhibitor cocktail (Roche). The lysates were clarified by centrifugation at 15,0006g for 15 min, and glutathione-agarose beads (Sigma) were used to bind the GST fusion proteins from the supernatant. GST protein-coated beads (4–8 mg) were incubated with pre-cleared in vitro translated proteins or human embryonic kidney (HEK) 293 T cell lysates Figure 1. Interaction of rEag1 N- and C-termini with 14-3-3h. (A) Schematic representation of (top) the structural topology of the rEag1 channel and (bottom) the rEag1 GST-N207 and GST-C0 fusion proteins. (B) Yeast two-hybrid assay. cDNA encoding rEag1-N207 or C0 segment was fused to the coding sequence for LexA DNA binding domain and subcloned into the pGilda vector. cDNA for the B42 transcriptional activation domain alone (Empty) or in combination with 14-3-3h was subcloned into the pJG4-5 vector. Yeasts co-transformed with the pGilda- and the pJG4-5- based plasmids were streaked on leucine-lacking plates. (C) GST pull-down assay of in vitro translated 14-3-3h. Pull-down products were immunoblotted with the anti-14-3-3h antibody. Indicated to the left are the molecular weight markers (in kDa). (D,E) Cell lysates prepared from HEK293T cells expressing myc-14-3-3h were used for GST pull-down assay with GST or the fusion protein GST-N207/GST-C0. (Left panels) Coomassie blue staining of the GST proteins. (Right panels) Immunoblotting of pull-down products with the anti-myc antibody. Input volume was 5% of that of the cell lysates for pull-down. doi:10.1371/journal.pone.0041203.g001 PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e41203