Fig. 3. Co-immunoprecipitation of new binding partners with TRIAD3.

a–e Flag-tagged candidate interactors WWP2 (a), RNF41 (b), AP2s1 (c), VPS35 (d), Ankrd13D (e) were co-overexpressed with HA-tagged TRIAD3B in HEK293T cells. HA-TRIAD3B was immunopurified and precipitates were analysed by western blotting using anti-flag antibody for detecting co-purified proteins (top panel) and anti-HA antibody for detecting immunopurified TRIAD3B (middle panel). Anti-flag lysate blots to illustrate expression levels of interactors are shown in the bottom panel (*, heavy and **, light chains of IP antibodies). f HRS interacts with TRIAD3 independent of ubiquitin chains. Immunoprecipitations were carried out as above. Additionally, catalytically inactive HA-TRIAD3B C745A was tested and co-precipitated flag-HRS similarly to the wt protein. Double point mutant UIM* (L269A, S270A) and UIM deletions mutant (ΔUIM) of HRS, which are defective in ubiquitin binding, also interacted with HA-TRIAD3B