14-3-3h Interacts with rEag1 Figure 3. The contribution of PAS and CNBHD to rEag1 interaction with 14-3-3h. GST pull-down assays of rEag1 N-terminal and C-terminal GST fusion proteins containing specific structural domains. (Upper panels) Schematic representation of the rEag1 N- terminal (A) or C-terminal (B,C) GST fusion proteins. (Lower left panels) Coomassie blue staining of the GST proteins. (Lower right panels) Immunoblotting of pull-down products with the anti-myc antibody. doi:10.1371/journal.pone.0041203.g003 Figure 2. Isoform specificity of 14-3-3 binding with rEag1 N- and C-termini. (A) GST pull-down assay of cell lysates from HEK293T cells transfected with various myc-tagged 14-3-3 isoforms. (Left panel) Coomassie blue staining of the GST proteins. (Right panel) Immuno- blotting of pull-down products with the anti-myc antibody. Input volume shown at the bottom corresponds to 5% of the total cell lysates for pull-down. (B) Quantification of the pull-down efficiency of different 14-3-3 isoforms. The protein band intensities of individual myc-14-3-3 isoforms affinity precipitated by GST-N207 or GST-C0 in (A) were divided by those of cognate total inputs, thereby minimizing the potential bias conferred by the variation in protein expression among different 14-3-3 isoforms. Densitometric scans of immunoblots were obtained from three independent experiments. doi:10.1371/journal.pone.0041203.g002 at 4uC overnight. The bead-protein complexes were then washed with buffer A [(in mM) 100 NaCl, 4 KCl, 2.5 EDTA, 20 NaHCO3, 20 Tris-HCl, pH 7.5, plus 1 PMSF, 1 Na3VO4, 1 NaF, 1 b-glycerophosphate] (with and without 1% Triton X-100), and the proteins were eluted by boiling for 5 min in the Laemmli sample buffer. In vitro protein translation was performed by using the TNT transcription-translation system (Promega). Cell culture and transient transfection Dissociated hippocampal culture and HEK293 cells stably expressing rEag1 were prepared as described previously [16]. HEK293 and HEK293T cells were maintained in DMEM (Invitrogen) supplemented with 2 mM L-glutamine, 100 units/ ml penicillin/streptomycin, and 10% (v/v) fetal bovine serum (Hyclone). One day before transfection, HEK cells were grown on poly-lysine-coated coverslips. DNA transfection was performed by using the Lipofectamine 2000 reagent (Invitrogen). PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e41203