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. Author manuscript; available in PMC: 2017 Feb 14.
Published in final edited form as: Nat Genet. 2010 Feb 28;42(4):303–312. doi: 10.1038/ng.538

Figure 6.

Figure 6

The VPS33B-VIPAR complex interacts with RAB11A. (a) Confocal fluorescence photomicrographs of HEK293 cells cotransfected with HA-VPS33B (not immunostained), with mCherry-VIPAR and with green fluorescent protein (GFP)-tagged RAB11A (GFP-RAB11A) showing colocalization (inset). Scale bar, 15 μm. (b) Confocal immunofluorescence photomicrographs of mIMCD-3 cells stained for endogenous Rab11a (green) and Vps33b (red). Nuclei are stained with TO-PRO-3. Scale bar, 15 μm. Colocalization of both markers is seen (inset). (c) Coimmunoprecipitation of endogenous Vps33b and Rab11a from mIMCD-3 cells after pulldown with Rab11a antibody or Vps33b antibody. Control (IgG molecules) failed to pull down the relevant protein. (d) HEK293 cells were cotransfected with HA-tagged VPS33B or empty vector, Myc-VIPAR or empty vector, and GFP-RAB11A. Coimmunoprecipitation experiments using HA-VPS33B or Myc-VIPAR as bait show that both VPS33B and VIPAR immunoprecipitate in the same complex as RAB11A. Overexpression of both HA-VPS33B and Myc-VIPAR was necessary for interaction with GFP-RAB11A to occur. Quantification of immunoprecipitation revealed that 6.7% (for HA; IP α HA) and 7.9% (for Myc; IP α Myc) of the RAB11A input was recovered. When GFP-tagged dominant negative (DN) RAB11A was used, no interaction between the VPS33B-VIPAR complex and GDP-locked dominant negative RAB11A could be seen.