Figure 5.

FTS and Hook proteins associate with class B and class C components of the HOPS complex. (A–E) Plasmids expressing the indicated HA-tagged Vps or FLAG-tagged Hook constructs were transfected into HEK293T cells and after 48 h, protein complexes were purified using the indicated anti-HA or anti-FLAG (M2) antibodies. Washed complexes and lysates as loading controls were subjected to SDS-PAGE and immunoblotting. (F–H) Plasmids expressing the indicated HA-tagged Vps or GST-tagged FTS constructs were transfected into HEK293T cells and after 48 h, protein complexes were purified using GSH-Sepharose. Washed complexes and lysates as loading controls were subjected to SDS-PAGE and immunoblotting. (I) Association of endogenous Hook1 with endogenous Vps18. HeLa cells were lysed in either buffer containing NP-40 or buffer containing CHAPS (see Materials and Methods), and cleared extracts were subjected to immunoprecipitation by using anti-Hook1 antibodies. Immune complexes were immunoblotted with either anti-Vps18 or anti-Hook1 antibodies.