2.4. Coimmunoprecipitation (CoIP) and In Vitro Assembly-Based CoIP

For CoIP analysis, 293T cells were seeded into 10 cm dishes with a density of 5 × 10⁶ cells and used for transient transfection with expression plasmids. Two to three days post transfection (d p.t.), CoIP was performed as described previously [26]. Antibody-coupled Dynabeads^TM Protein A (10002D, Thermo Fisher Scientific) were used to obtain specific immunoprecipitates and CoIP samples were further analyzed by Western blot (Wb). For CoIPs performed on the basis of protein complexes formed by in vitro assembly, 293T were singly transfected for transient expression using plasmids coding for proteins carrying HA and Flag tags, respectively. One of the two proteins was immunoprecipitated with the tag-specific antibody in an initial IP for 1–2 h. After washing, the sample containing the second tagged protein, or alternatively endogenously expressed untagged protein, was added for assembly and a final CoIP was performed for 1.5–2 h or overnight.