JCS01542 Supplementary Material

Files in this Data Supplement:

• Supplementary Materials and Methods (PDF)
• Supplemental Figure 1 -

  Fig. S1. Clustal W alignment of the hypervariable domain of Rab5 proteins from different eukaryotic species. hRab5a, hRab5b and hRab5c are from human; Ypt51, Ypt52 and Ypt53 are from Saccharomyces cerevisiae and TbRab5A and TbRab5B are from T. brucei. The position of the conserved guanine nucleotide-binding SAK motif is underlined in black, and the positions of the a5 helix, the hypervariable domain and the geranylgeranylation (GG) motif are indicated at the top of the diagram. Residues identical in all Rab5 homologues are shown in red, variant residues in black. Green and blue indicate conservative and less conservative substitutions, respectively.

• Supplemental Figure 2 -

  Fig. S2. Triton X-114 partitioning of Rab proteins in HEK 293 cells. HEK 293 cells were transfected with the EGFP-tagged form of the indicated Rab (A and B), Rab hybrid (A) or Rab mutant (B). 24 hour post-transfection, cells were harvested and processed for Triton X-114 partitioning as described previously (Gomes et al., 2003). An equivalent amount of the aqueous (A) and detergent (D) phases were fractionated using 12% SDS-PAGE gels, transferred onto PVDF membrane and probed with anti-GFP polyclonal antibodies (Abcam, UK).

• Supplemental Figure 3 -

  Fig. S3. Localisation of myc-tagged Rab proteins in AtT20 (A-L) or HeLa cells (M-R). AtT20 or HeLa cells were transfected with the myc-tagged forms of the indicated Rab or Rab hybrid proteins and analysed by confocal microscopy as described under Materials and Methods using anti-myc polyclonal antibodies (1:200; Upstate, USA).