Cell lines were collected and stored at − 80 °C until use. SDS-PAGE and western blotting were done as described previously33 (link). The anti-Flag antibody (clone M2, Sigma-Aldrich Crop., St. Louis, MO) and the anti-actin mouse monoclonal antibody (clone AC-15, Sigma-Aldrich Corp.) were used as primary antibodies. The horseradish peroxidase (HRP)-conjugated secondary anti-rat (Zymed, San Francisco, CA) and anti-mouse (Bio-rad Laboratories, Inc.) antibodies were used as secondary antibodies. The membrane was visualized by immersion in Western Lightning chemiluminescence reagent (Perkin Elmer, Foster City, CA, USA). Immunoreactive bands were visualized using the Luminescent Image Analyzer LAS 3,000 Mini instrument (FIJIFILM, Tokyo, Japan).
For immunoprecipitation, clarified cell lysates were pre-cleared with 10 µl of protein A/G agarose (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at 4 °C with rotation. The resultant supernatant was mixed at 4 °C overnight with 1 µg of each antibody, which was premixed with 10 µl of protein A/G agarose for 1 h at 4 °C. The immunoprecipitates were washed three times with PBS and used for Western blotting as described above.
For immunoprecipitation, clarified cell lysates were pre-cleared with 10 µl of protein A/G agarose (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at 4 °C with rotation. The resultant supernatant was mixed at 4 °C overnight with 1 µg of each antibody, which was premixed with 10 µl of protein A/G agarose for 1 h at 4 °C. The immunoprecipitates were washed three times with PBS and used for Western blotting as described above.