Anti flag m5 monoclonal antibody

Samples were boiled with SDS sample buffer [150 mM Tris-HCl pH 6.8, 0.6% SDS, 15% glycerol, 0.009 mg/μl Bromophenol blue, 5% 2-mercaptoethanol and 1 unit Complete Mini (Roche)] for 5 min, and loaded on 12% polyacrylamide gel followed by transfer onto PVDF membrane (GE Healthcare). Anti-FLAG M5 monoclonal antibody (1:1,000; Sigma) was used for primary antibody and ECL Peroxidase labeled anti-mouse antibody (1:10,000; GE Healthcare) was used for secondary antibody. The band was detected by ECL Ultra Lumigen TMA-6 (GE Healthcare) and Ez-Capture MG (ATTO).

Immunoprecipitation assays were performed as previously described [29 (link)]. Briefly, HEK293FT cells were co-transfected with vectors expressing a His-tagged GMEB1 protein (GMEB1-His) (1 μg) together with a FLAG-tagged TRAF3 protein (TRAF3 wt-flag) (0.5 μg) or the FLAG-tagged mutant forms of TRAF3, TRAF3 M7 (TRAF3 M7-flag) (0.5 μg) or TRAF3ΔC-flag (0.5 μg), using the calcium phosphate transfection method. The cells were harvested approximately 20 h post transfection and lysed in 0.5% NP40 lysis buffer [25 mM Tris–HCL pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Glycerol, 0.5% NP-40, 1 mM DTT, 1 mM PMSF 100 mM, 1 × protease inhibitor cocktail (Roche, Basel, Switzerland)]. His-tagged proteins (GMEB1-His) were immunoprecipitated with Ni–NTA beads (Qiagen, Hilden, Germany). The immunoprecipitated complex (IP) or total cell lysates were analyzed by SDS-PAGE on a 7.5–8% gel and subjected to immunoblot analysis with the anti-His rabbit polyclonal antibody His probe (Santa Cruz Biotechnology, Santa Cruz, USA), recognizing GMEB1 (80 kDa) or the anti-FLAG monoclonal antibody M5 (Sigma-Aldrich, St. Louis, USA), recognizing both TRAF3 wt (58 kDa) and TRAF3 M7 (58 kDa). The LMP1 monoclonal antibody OT22CN, which recognizes the amino-terminus of LMP1, was used against LMP1(1–231), and the anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, USA) was used against β-actin.