Fig. 5. Golgi targeting of INPP5B requires RAB binding. (A) Sequence alignment of the OCRL1 and INPP5B linker regions showing the conservation of residues whose mutation in OCRL1 reduces RAB binding and membrane targeting (red arrows). (B) Beads containing GST-RAB5Q79L or RAB6Q72L were incubated with buffer alone or HeLa cell extract containing full-length wild-type GFP-INPP5B or the indicated point mutants and bound protein detected by western blotting with antibodies against GFP. (C) Localisation of the indicated GFP-tagged INPP5B proteins (green) in HeLa cells double-labelled with antibodies to GM130 (red). Bar, 10 µm. (D) The indicated GFP-tagged INPP5B proteins were expressed in metabolically labelled HeLa cells and immunoprecipitated with antibodies against GFP before limited digestion with the indicated amounts of trypsin. Digested proteins were analysed by SDS-PAGE and autoradiography. Markers indicate the molecular mass (kDa).