Additional Supporting Information may be found in the online version of this article.

HEP_25523_sm_SuppFig1.tif21123KSupporting Information Figure 1. Effect of co-transfection with Rab5a and Rab5a DN. TacCterm was co-transfected in HEK292T cells with Rab5a or Rab5a DN for 18-24 hrs. (A) Cells were lysed in RIPA buffer and lysate separated by PAGE and TacCterm detected by anti-Tac antibody. Densitometry results from 3 experiments and a representative blot is shown. Slightly less TacCterm was detected after co-transfection with Rab5a, but not Rab5a DN. NS, Not significant, student two tailed t-test. (B) Cell ELISA was performed as described in Methods. Co-transfection of TacCterm and Rab5a resulted in slightly, but not significantly, less surface expression of TacCterm. N=3. NS, Not significant, student two tailed t-test. (C) There was no significant difference in internalization of TacCterm after co-transfection with Rab5a or Rab5a DN, although there was a tendency for more internalization with Rab5a and less with Rab5a DN. This suggests that endocytosis of TacCterm may involve a Rab5a and clathrin dependent pathway, as well as other possible pathways.
HEP_25523_sm_SuppFig2.tif4441KSupporting Information Figure 2. Rate of internalization of full length, human BSEP in HEK293T cells. Forty-eight hours after transfection with WT GFP-BSEP, cells were cooled and surface biotinylation was performed. The cells were warmed to 37°C for the indicated time to allow for endocytosis, then cooled and remaining surface biotinylated protein stripped before the cells were lysed. Endocytosed BSEP was cleared from the lysate by incubation with Streptavidin agarose resin and separated by PAGE. Protein bands detected with anti-GFP were quantitated and expressed as a percentage of the total GFP-BSEP in cells with no stripping. Data are plotted against time of endocytosis to give a rate of endocytosis/min, which is given in parenthesis above the data point. These data are similar to the endocytic rates obtained for the TacCterm construct, confirming the importance of the C-terminal motif in BSEP. N=3
HEP_25523_sm_SuppFig3.tif1914KSupporting Information Figure 3. Sequence alignment of the C-termini of other ABCB subfamily members. Note the conservation of the Yxxphi and dileucine motifs in bold, suggesting that members of the closely related ABCB subfamily may also be regulated by these motifs.

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