14-3-3h Interacts with rEag1 Figure 4. Phosphorylation-independent interaction of rEag1 with 14-3-3h. (A) Co-immunoprecipitation of myc-14-3-3h and rEag1 proteins. (Left panel) rEag1/rEag2 was co-expressed with an empty vector (2) or myc-tagged 14-3-3h (+) in HEK293T cells. Cell lysates were immunoprecipitated (IP) by using the anti-myc antibody, followed by immunoblotting (WB) with the anti-myc or the anti-rEag1/rEag2 antibody. The protein bands corresponding to rEag1/rEag2 and 14-3-3h are highlighted with arrow and arrowhead, respectively. (Right panel) Cell lysates from myc-14-3-3h only or co-expression of rEag1 and myc-14-3-3h were immunoprecipitated by using the anti-rEag1 antibody. Input volumes correspond to 5% of the total cell lysates used for immunoprecipitation. These co-immunoprecipitation data are representative of three to five independent experiments. (B) rEag1 was co-expressed with an empty vector or myc-tagged 14-3-3h in HEK293T cells. 24 hrs after transfection, indicated cells were subject to 1-hr treatment with 1 mM okadaic acid or staurosporine. (Upper panel) Total cell lysates were immunoblotted with the anti-Akt (total Akt) or anti-phosphorylated Akt (pAkt) antibodies to monitor the cellular phosphorylation status. b-actin was run as a loading control. (Lower panel) Cell lysates were immunoprecipitated (IP) by using the anti-myc antibody, followed by immunoblotting (WB) with the anti-myc or the anti-rEag1 antibody. (C) Quantification of (upper panel) the Akt phosphorylation level (pAkt/Akt) and (lower panel) the co-immunoprecipitation (CO-IP) efficiency of 14-3-3h and rEag1. The CO-IP efficiency was determined by the ratio of the protein band intensities of immunoprecipitated rEag1 to those of cognate total inputs. The mean values were subsequently normalized with respect to that of the no-treatment control of 14-3-3h/rEag1 co- expression. Densitometric scans of immunoblots were obtained from three independent experiments. Asterisk denotes a significant difference from the no-treatment control of 14-3-3h/rEag1 co-expression (*, t-test: p,0.05). doi:10.1371/journal.pone.0041203.g004 PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e41203