Anti myc or anti flag antibody

To detect interactions among the proteins, plasmids were transfected into COS7 cells (5 × 10⁵ cells/6 cm dish) using PEI. Forty hours after the transfection, cells were lysed in 500 μL of TNE buffer (10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM ethylenediamine-N′,N′,N′,N′-tetraacetic acid (EDTA), 1% NP-40, 1 mM phenylmethylsulfonyl-l-fluoride (PMSF), 5 μg/mL leupeptin, 100 U/mL aprotinin, 2 mM sodium vanadate, 40 mM NaF, and 20 mM β-glycerophosphate). The cell lysates were precleared with protein G-Sepharose beads (GE Healthcare) for 30 min at 4°C and then incubated with anti-Myc or anti-Flag antibody (Sigma) for 2 h at 4°C. The protein complexes were immunoprecipitated by incubation with protein G-Sepharose beads for 30 min at 4°C followed by three washes with TNE buffer. The immunoprecipitated proteins and aliquots of the total cell lysates were boiled for 5 min in sample buffer, separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a Hybond-C Extra membrane (GE Healthcare). The membranes were probed with primary antibodies. The primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies and chemiluminescent substrate (Thermo Scientific). Protein expression in the total cell lysates was evaluated by Western blot analysis.

A total of 400 µl of cell lysate was incubated overnight at 4 °C with 2 µg of anti-Myc or anti-Flag antibody (Sigma).Protein A-Sepharose (GE) was washed three times with lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% NP-40) and centrifuged for 30 s at 1000 rpm. An ~40 µl suspension of protein A-Sepharose was subjected to precipitation of the immunocomplex. After incubation at 4 °C for 2 hr, beads were washed three times with lysis buffer and proteins were eluted from the beads by boiling for 10 min, and were then subjected to Western blotting.