Nudel binds to HSPC300 tightly and facilitates the HSPC300-WAVE2 interaction. (A) A diagram summarizing the experiments and results in B. For simplicity, only representative complexes are displayed to assist understanding. (B) The Nudel-HSPC300 interaction was stable. HEK293T cells transfected as indicated to overexpress HA-HSPC300 and/or Flag-Nudel were lysed, mixed with GST-HSPC300 purified from E. coli, and subjected to the first round of co-IP (IP1) using the anti-HA resin (lanes 1-3). The immunoprecipitates eluted with the HA peptide were then subjected to the second round of co-IP (IP2) using the anti-Flag resin (lanes 4 and 5) or to the GST pull-down (lane 6) as indicated. (C) Nudel repressed the formation of the hybrid HSPC300 oligomer in a dose-dependent manner. HEK293T cell lysate containing HA-HSPC300 was mixed with purified GST, GST-HSPC, and Flag-Nudel as indicated, followed by the GST pull-down. Flag-Nudel was affinity-purified from HEK293T cell lysate using the anti-Flag resin and eluted with the Flag peptide. (D) Nudel promoted the WAVE2-HSPC300 interaction. HEK293T cells transfected with indicated amount of plasmids were lysed and subjected to co-IP with the anti-Flag resin.