Coimmunoprecipititation and Immunoblotting
HEK293 cells were transfected with plasmids encoding myc- and FLAG-tagged cDNA constructs using Gene Juice (Novagen) at approximately 0.1 μg DNA per ml of media. Within 24 hr, cells were collected in 1× PBS and lysed in 1 ml of the following buffer: 50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1% IGEPAL, 0.5 mM EDTA, and 10% glycerol supplemented with protease (Roche Complete) and phosphatase inhibitors (Cocktails 2 and 3, Sigma Aldrich). Lysates were centrifuged at 16,000 × g for 30 min at 4°C. Approximately 2 mg of each lysate was precleared with 4 μg of rabbit control IgG antibody for 2 hr at 4°C and then incubated with MagSi/protein A beads (MagnaMedics, Germany) for 1 hr. Lysates were then incubated with either 2 μg of rabbit anti-CCDC114 or anti-CCDC151 antibody and 2 μg of control IgG antibody overnight at 4°C, and then incubated with MagSi/protein A beads for 1 hr to capture immunoprecipitates. Bead complexes were washed four times in lysis buffer and then resuspended in 1× LDS buffer supplemented with DTT and heated for 10 min at 90°C. Lysates were electrophoresed in NuPAGE 4%–12% Bis-Tris gels, transferred to PVDF filters, and subsequently immunoblotted with either anti-myc (A.7) or anti-FLAG (M2) mouse monoclonal antibodies. PVDF filters were washed three times in TBS-T (10 min each) before blocking in 5% BSA for 2 hr at room temperature. Filters were then washed three times (10 min each) before incubation with primary antibody (diluted in TBS-T) overnight at 4°C. Filters were washed three times (10 min each) and then incubated with goat anti-mouse HRP secondary antibody for 1 hr at room temperature. Filters were then washed four times and developed by ECL using Prime Western Blotting Detection Reagent (Amersham). Images were digitally acquired using a FUSION-SL Advance Imager (PeqLab) and modified for contrast using Adobe Photoshop v.CS4. All wash and incubation steps were performed with gentle shaking. The following antibodies were used: rabbit polyclonal anti-CCDC151 (Atlas Antibodies, HPA044184), rabbit polyclonal anti-CCDC114 (Atlas Antibodies, HPA042524), mouse monoclonal anti-myc (1:2,000; clone A.7, Abcam), rabbit polyclonal control IgG (sc-2027, Santa Cruz), mouse monoclonal anti-FLAG (1:2,000; clone M2, Sigma Aldrich), and goat anti-mouse HRP antibody (1:5,000; NA931V, GE Healthcare).