The Co-IP procedure was performed as described previously 28 (link). Briefly, different combinations of plasmids were cotransfected into cells, followed by incubation at 37°C for 48 h. The transfected cells were then lysed in 1×RIPA lysis buffer supplemented with a protease inhibitor cocktail. After centrifuging at 13,000 rpm for 15 min, the supernatant was incubated with anti-Flag agarose beads and anti-c-Myc agarose beads (Sigma-Aldrich, #A7470) at 4°C for 2 h. The beads were then washed 5 times with 1×RIPA lysis buffer, and protein interactions were determined using immunoblot assays.
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