Mouse monoclonal anti myc

HEK-Nav1.6 cells were transfected with FGF14-6xmyc, and incubated with either DMSO (0.5%) or ZL181 (50 μM). Cells were then washed with cold PBS. Subsequently, 50 μL of lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1% NP-40) and 1 μL Protease inhibitor cocktail (set #3, Calbiochem, Billerica, MA) were added. Cell extracts were collected, sonicated for 20 s, and centrifuged at 4 °C, 13,000 g for 15 min, and 2× sample buffer containing 50 mM tris (2-carboxyethyl) phosphine (TCEP) was added. Mixtures were heated for 10 min at 55 °C and resolved on 4–15% polyacrylamide gels (BioRad, Hercules, CA). Resolved proteins were transferred to PVDF membranes (Millipore, Bedford, MA) for 2 h at 4 °C and blocked in tris-buffered saline (TBS) with 3% nonfat dry milk and 0.1% Tween-20. Membranes were then incubated in blocking buffer containing mouse monoclonal anti-Myc (1:1000; 9E10 clone Santa Cruz Biotechnology) or mouse monoclonal anti-PanNav channel (1:1000; Sigma) antibody overnight. Washed membranes were incubated with goat anti-mouse HRP antibody (1:5000–10,000; Vector Laboratories, Burlingame, CA) and detected with ECL Advance Western blotting Detection kit (GE Healthcare). Subsequently, protein bands were visualized using FluorChem HD2 System and analyzed with AlphaView 3.1 software (ProteinSimple, Santa Clara, CA).

Standard Western blotting method from our published protocol was used with minor modifications (Kamiya et al., 2005 ; Kamiya et al., 2006 (link)). Protein samples were analyzed by SDS-PAGE and Western blotting with the following antibodies: rabbit anti-DISC1 polyclonal antibody (1:500, ECM Biosciences), mouse anti-GAPDH monoclonal antibody (1:10,000, Santa Cruz), mouse anti-β-tubulin monoclonal antibody (1:5000, Sigma-Aldrich). For co-immunoprecipitation, we used rabbit polyclonal anti-HA (10ug, Clontech) and mouse monoclonal anti-myc (5ug, SantaCruz) antibodies. Species-appropriate secondary antibodies were conjugated to HRP (Pierce) and detected by enhanced chemiluminescence (Thermo Scientific) using ImageQuant LAS4000 mini (GE Healthcare). Quantification of immunoblot was performed with ImageJ64 (National Institutes of Health). Optical density of immunoreactivity in Western blotting was acquired with Image J software (http://rsb.info.nih.gov/ij/). Protein levels were normalized to GAPDH and expressed as relative % integrated intensity.