14-3-3h Interacts with rEag1 Figure 6. Localization of 14-3-3h and rEag1 in synaptosomal and PSD fractions. (A) Subcellular fractionation separated rat brains into multiple fractions: homogenate (H), soluble fraction (S1), crude membrane fraction (P2), synaptosomal fraction (SPM), and two postsynaptic density (PSD) preparations (PSD I: one Triton X-100 wash; PSD II: two Triton X-100 washes), all of which were subject to immunoblotting analyses with the indicated antibodies. 25 mg and 5 mg refer to the amount of total protein loaded in each lane. (B) Quantitative analyses of protein abundance in different subcellular fractions. Densitometric scans of immunoblots were obtained from three to five independent experiments. Data were presented as normalized values with respect to cognate protein expression levels in the homogenate (H) fraction. doi:10.1371/journal.pone.0041203.g006 extracted in ice-cold 0.5% Triton X-100/50 mM Tris-HCl (pH 7.9) for 15 min and centrifuged at 32,0006g for 45 min to obtain the PSD I pellet. The pellet was resuspended and further extracted a second time with 0.5% Triton X-100/50 mM Tris- HCl (pH 7.9), followed by centrifugation at 200,0006g for 45 min to obtain the PSD II pellet. Protein concentration was determined by the Bio-Rad protein assay kit (Bio-Rad). Electrophysiology Conventional whole-cell patch clamp technique was used to record rEag1 K+ currents as described previously [17]. In brief, whole-cell patch clamp was performed 24–48 hrs post-transfection of HEK293 or HEK293T cells. Patch electrodes were filled with a solution containing (in mM) 140 KCl, 1 MgCl2, 10 EGTA, 10 HEPES, pH 7.2. External recording solution comprised (in mM) 140 NaCl, 5 KCl, 1 CaCl2, and 10 HEPES, pH 7.2. Data were acquired with an Axopatch 200A amplifier (Molecular Devices) and digitized with the Digidata 1322A system and the pCLAMP 9.0 software (Molecular Devices). Cells with large currents in which voltage clamp errors might appear were excluded from data analyses. cRNA injection and conventional two-electrode voltage clamp recording of rEag1 K+ currents in Xenopus oocytes were performed as described previously [17]. In brief, stage V–VI oocytes were selected for cRNA injection. For all cRNA injection paradigms, the total volume of injection was always 41.4 nl per oocyte and the final injection amount for rEag1 cRNA was about 5 ng per oocyte. 2–3 days after cRNA injection, oocytes were functionally assayed in a recording bath containing the Ringer solution [(in mM): 115 NaCl, 3 KCl, 1.8 CaCl2, 10 HEPES, pH 7.2]. Voltage-clamp protocols in oocytes were applied with an OC-725C oocyte clamp (Warner) and data were digitized with the Digidata 1320A system and the pCLAMP 8.2 software (Molecular Devices). Passive membrane properties were compensated by using the -P/4 leak subtraction method provided by the pCLAMP software. Data were sampled at 10 kHz and filtered at 1 kHz. All recordings were performed at room temperature (20–22uC). Non-stationary fluctuation analyses [18] were employed to estimate the single channel conductance of rEag1 channel from whole-cell currents (series of 40–100 traces) evoked by 300-ms depolarization from 290 mV to +40 mV. The mean (I) and variance (s2) were calculated and plotted against each other, followed by curve fitting with the parabolic function: s2 = iI2I2/N, where i and N represents the single channel current and the total number of channels, respectively. The estimated i derived from curve fitting was in turn used to calculate the single channel conductance, based on the theoretical assumption that the K+ equilibrium potential was 284 mV. Statistic analyses All values were presented as mean 6 SEM. The significance of the difference between two means was tested by Student’s t-test, whereas means from multiple groups were compared by one-way ANOVA. All statistical analyses and curve fitting were performed with the Origin 7.0 software (Microcal Software). Results Interaction of 14-3-3h with the N- and C-termini of rEag1 in vitro We carried out the yeast two-hybrid screening of a rat brain cDNA library by using the N-terminus (amino acids 1–207) of the rEag1 protein (rEag1-N207) as the bait (Fig. 1A). One of the positive clones isolated by the screening was 14-3-3h. 14-3-3 proteins are ubiquitously expressed in all eukaryotes [19]. In PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e41203