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. 2006 Aug 10;25(16):3750–3761. doi: 10.1038/sj.emboj.7601274

Figure 5.

Figure 5

Rab binding-deficient OCRL1 mutants fail to target to the Golgi apparatus and endosomes. (A) Cell lysates prepared from HeLaM cells expressing GFP-tagged full-length OCRL1 or the indicated point mutants were incubated with beads containing either GST alone, GST-clathrin terminal domain (TD), or GST-rab6Q72L, and bound proteins analysed by Western blotting with antibodies to OCRL1. (B) Cell lysates were incubated with beads containing GST alone, GST-rab1Q70L, GST-rab5Q79L, or GST-rab6Q72L and bound proteins analysed by Western blotting with antibodies to OCRL1. (C) HeLaM cells expressing GFP-tagged full-length OCRL1 or the indicated point mutants were analysed by immunofluorescence microscopy with antibodies to GalNacT2. (D) HeLaM cells were cotransfected with myc-rab5Q79L and GFP-tagged full-length OCRL1 or the indicated point mutants were analysed by immunofluorescence microscopy with antibodies to myc. Bar, 10 μm. (E, F) Quantitation of Golgi and endosomal targeting of WT and mutant GFP-OCRL1. Targeting was defined as a discernable increase in GFP fluorescence above background on GalNacT2 (E) or Myc-Rab5Q79L (F) positive structures. The results are expressed as the mean±s.d. from three independent experiments, counting 100 cells per experiment.