FYCO1 is a novel LC3B-interacting protein. (A and B) Endogenous FYCO1 binds to GST-LC3B. GST-LC3B bound to glutathione–Sepharose beads was incubated with HeLa cell lysate, and copurified proteins were identified by Coomassie blue staining and mass spectrometry (A) or immunoblotting with anti-FYCO1 antibody (B). (C) GST-LC3B but not GST-p62 or GST alone binds to FYCO1 in a GST pull-down assay. GST, GST-LC3B, or GST-p62 bound to glutathione–Sepharose beads was incubated with [³⁵S]methionine-labeled myc-FYCO1. Formed protein complexes were isolated and visualized by autoradiography (top) or Coomassie blue staining (bottom). (D) GFP-LC3B immunoprecipitates endogenous FYCO1 from HeLa cell lysate. HeLa cells expressing GFP or GFP-LC3B were lysed and processed as in B. IP, immunoprecipitation; MM, molecular mass; WB, Western blot.