To study the interaction between 2B and VDAC3, we transfected Vero cells with vectors encoding GFP-2B-Myc and N-FLAG-VDAC3. Immunoprecipitation was carried out with Dynabead Protein G magnetic beads that had been pre-coated with Myc monoclonal antibody (9E10) or FLAG M2 monoclonal antibody (Sigma-Aldrich, St. Louis, MI, USA) as described elsewhere [7 (link)].
For analysis of the immunoprecipitated proteins, the protein-bound beads were mixed with Laemmli sample buffer and heated at 95 °C for 5 min. The sample was analyzed by Western blotting as previously described [38 (link)]. The antibody to actin (Clone AC-40) was purchased from Sigma-Aldrich. A horseradish peroxidase (HRP)-conjugated secondary antibody to mouse IgG (SC-2005) was available from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). The HRP-conjugated anti-Myc antibody (R951-25) and anti-FLAG antibody (MA1-91878) were purchased from Thermo Fisher Scientific.
For analysis of the immunoprecipitated proteins, the protein-bound beads were mixed with Laemmli sample buffer and heated at 95 °C for 5 min. The sample was analyzed by Western blotting as previously described [38 (link)]. The antibody to actin (Clone AC-40) was purchased from Sigma-Aldrich. A horseradish peroxidase (HRP)-conjugated secondary antibody to mouse IgG (SC-2005) was available from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). The HRP-conjugated anti-Myc antibody (R951-25) and anti-FLAG antibody (MA1-91878) were purchased from Thermo Fisher Scientific.