Figure 5. Endogenous expression of 14-3-3h and rEag1 in neurons. (A) Co-immunoprecipitation of 14-3-3h and rEag1. Detergent solubilized proteins from the lysates of rat forebrain were immunopre- cipitated (IP) with the anti-14-3-3h (upper panel) or the anti-rEag1 antibody (lower panel), followed by immunoblotting (WB) analyses with the anti-14-3-3h or the anti-rEag1 antibody. The non-immune mouse or rabbit IgG was used in parallel as negative control. Input volumes correspond to 5% of the total cell lysates used for immunoprecipitation. The arrowhead and arrow refers to the protein bands of 14-3-3h and rEag1, respectively. (B) Immunofluorescence staining of rEag1 (left panels) and 14-3-3h (middle panels) in cultured hippocampal neurons. The area highlighted in the white boxes is viewed under a higher magnification (I, II). Arrows label the sites of co-localization of 14-3-3h and rEag1 (right panels), which displayed significant punctuate patterns over a wide region along the neurites. Scale bar, 25 mm. These co- immunoprecipitation and immunofluorescence data are representative of four to seven independent experiments. doi:10.1371/journal.pone.0041203.g005 Biotinylation of cell surface proteins Cell surface biotinylation and streptoavidin pull-down was performed as described previously [17]. In brief, cells were incubated in 1 mg/ml sulfo-NHS-LC-biotin (Thermo Scientific) at 4uC for 30 min with gentle rocking. After termination and solubilization, insolubilized material was removed by centrifuga- tion and the solubilized cell lysates were incubated for 16 hrs at 4uC with streptavidin-agarose beads (Thermo Scientific). The 14-3-3h Interacts with rEag1 biotin-streptavidin complexes were eluted from the beads by boiling for 5 min in the Laemmli sample buffer. Co-immunoprecipitation and immunoblotting Cell lysates were prepared by solubilizing cells in the buffer A containing 1% Triton X-100 and protease inhibitor cocktail (Roche). Insolubilized materials were removed by centrifugation. Solubilized HEK293T cell lysates or brain homogenates were pre- cleared with protein-G beads and then incubated for 16 hrs at 4uC with protein G-Sepharose (GE Healthcare Biosciences) previously coated with the indicated antibodies. After washing with ice-cold lysis buffer, the immune complexes were eluted from beads by boiling in the Laemmli sample buffer. Proteins were then separated on SDS-PAGE, immunoblotted with appropriate di- lution of primary antibodies, and visualized with the ECL detection system (Western Lightning Detection Kit, PerkinElmer). Immunoblots were scanned, and protein signals were quantified by using the ImageQuant software (GE Healthcare Biosciences). The antibodies used in this study include anti-14-3-3h and anti- pan 14-3-3 (Santa Cruz Biotechnology); anti-b-actin (Sigma); anti- Akt and anti-pAkt (Cell Signaling); anti-GFP (Abcam); anti-myc (clone 9E10); anti-PSD-95 (Affinity BioReagents); anti-rEag1, anti- rEag2, and anti-Herg (Alomone Labs); and anti-synaptophysin [16]. Immunofluorescence microscopy Immunofluorescence staining was performed as described [16,17]. In brief, after fixation, permeabilization, and blocking, cells were incubated overnight at 4uC in appropriate dilutions of primary antibodies (rabbit anti-rEag1 or mouse anti-14-3-3h antibody), followed by incubation with secondary antibodies [Alexa Fluor 568 goat-anti-mouse or Alexa Fluor 488 goat-anti- rabbit antibodies (Invitrogen Molecular Probes)] at 1:500 dilution for 1 hr at room temperature. Nuclei were labeled with DAPI. After final washes and mounting, fluorescence images of the fixed cultures were viewed with a fluorescence laser-scanning confocal microscope (Leica). Preparation of rat brain homogenates Rat brain tissues were homogenized with a motor driven glass- Teflon homogenizer in ice-cold dissociation buffer [(in mM) 320 sucrose, 1 MgCl2, 0.5 CaCl2, 1 NaHCO3, 1 PMSF and 1 mg/l leupeptin) and the cell debris was removed by centrifugation at 1,4006g for 10 min. The supernatant was saved, and the pellet was resuspended by homogenization in ice-cold dissociation buffer and pelleted again. The remaining pellet was discarded and the combined supernatants were pelleted (13,8006g for 10 min) again. The final pellet was resuspended in the buffer A containing 1% Triton X-100 and protease inhibitor cocktail. Subcellular fractionation of rat brains Adult rat forebrains were homogenized in buffer H1 [(in mM) 320 sucrose, 1 NaHCO3, 0.5 CaCl2, 0.1 PMSF] containing a cocktail of protease inhibitors (Roche) and centrifuged at 1,4006g for 10 min to recover the supernatant S1 and the pellet P1. S1 fraction was subject to centrifugation at 13,8006g for 10 min to obtain the P2 pellet. The pellet was resuspended in buffer H2 [(in mM) 0.32 M sucrose and 1 mM NaHCO3)] and layered onto the top of the discontinuous sucrose density gradient by using 0.85, 1.0, and 1.2 M sucrose layers. The gradient was centrifuged at 65,0006g for 2 hrs in a Beckman Instruments SW- 28 rotor and the synaptosomal fraction was recovered from the 1.0–1.2 M sucrose interface. The synaptosomal fraction was PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e41203