14-3-3h Interacts with rEag1 Figure 10. Estimation of the single channel conductance of rEag1 channel under different co-expression paradigms. rEag1 K+ channels were co-expressed with either the control vector, difopein, or 14-3-3h in HEK293 cells. (A) Comparison of the mean current density (pA/pF) at +40 mV: rEag1+vector, 111.269.5; rEag1+difopein, 126.9613.2; rEag1+14-3-3h, 76.865.8. The numbers in the parentheses refer to the number of cells analyzed, and the asterisk denotes significant difference from the rEag1+vector control (*, t-test: p,0.05). (B) Representative non-stationary fluctuation analysis of rEag1 K+ currents. (Top row) Whole cell current traces were evoked by 300-ms depolarizations from 290 mV to +40 mV, which were used to generate the ensemble mean (second row) and variance (third row). (Bottom row) The mean-variance plot (during the 300-ms test pulse, with the linear capacitative component eliminated)(open circles) was fit with a parabolic function (solid curve) to estimate the single channel current (0.9 pA). (C) Box plot presentation of the distribution of the estimated single channel conductance. Mean values (pS): rEag1+vector, 7.560.9; rEag1+difopein, 7.062.3; rEag1+14-3-3h, 8.061.1. No significant difference was found among the three co-expression conditions (One-way ANOVA: p.0.05). doi:10.1371/journal.pone.0041203.g010 4C, neither okadaic acid nor staurosporine treatments led to noticeable change in the 14-3-3h-binding efficiency of rEag1, consistent with idea that 14-3-3h may interact with rEag1 in a phosphorylation-independent manner. Given that both 14-3-3h and rEag1 proteins are abundantly expressed in the brain, it is imperative to determine whether the potential interaction of these two proteins can also be verified in neurons. Crude membrane (P2) fractions prepared from rat forebrain homogenates were subject to immunoprecipitation with the anti-14-3-3h antibody, followed by immunoblotting with the anti-rEag1 antibody. As demonstrated in Figure 5A, rEag1 was effectively co-immunoprecipitated with 14-3-3h, suggesting that in the rat brain, endogenous 14-3-3h and rEag1 co-existed in the same protein complex. Next we asked if 14-3-3h and rEag1 shared overlapping subcellular localization in neurons. Confocal microscopic analyses of cultured rat hippocampal neurons co-immunostained with the anti-14-3-3h and anti-rEag1 antibodies showed that the immunor- eactivities of the two proteins exhibited an extensive subcellular co- localization pattern over a broad region encompassing the soma and neurites (Fig. 5B). In addition, the rEag1 immunofluorescence displayed a notable punctate pattern over the neurites, in agreement with our previous report that rEag1 proteins may be present in the synaptic region [16]. Interestingly, in the neurites PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e41203