SMN interacts with the Ub ligase Itch. (A) Empty pRK7 vector (mock), FLAG-SMN or FLAG-SMNΔ7 was transfected into 293T cells. Expressed SMN or SMNΔ7 was immunoprecipitated with an anti-FLAG antibody, followed by immunoblotting of FLAG, Itch and Huwe1. (B) Endogenous SMN in 293T cells was immunoprecipitated with an anti-SMN antibody, followed by immunoblotting of SMN, Gemin 8 and Itch. (C) Endogenous Itch in 293T cells was immunoprecipitated with an anti-Itch antibody, followed by immunoblotting of SMN, Gemin 8, Gemin 3 and Itch. (D) Recombinant GST or GST-Itch was incubated with recombinant SMN or SMNΔ7 for in vitro GST pull-down assays. Proteins bound on glutathione resin were applied for immunoblotting of SMN or visualized by ponceau S staining.