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Fig. 4. Characterization of Rabip4 subcellular localization by immunofluorescence studies. (A) Subcellular localization of Rabip4 compared with organelle markers. 3T3-L1 adipocytes were processed for indirect immunofluorescence using antibodies to Rabip4 (a,a',d), together with anti EEA1 mAb (b,b') or anti-TfR mAb (e). Panels c,c', and f show the corresponding merged images. One confocal section taken in the middle of an adipocyte is shown for each labeling. a'-c' are enlarged views of the boxed regions in a-c. Arrowheads indicate vesicles containing juxtaposed EEA1 and Rabip4 labeling. The insets in panels d-f correspond to an enlarged view of the perinuclear region. In panels g,h,i, TGN-38 localization (shown in h) was analyzed versus GFP-Rabip4 (shown in g). Panel i shows the merge images. (B) Rabip4 is mainly colocalized with Rab4, but not Rab5, Rab11, or Rab7. 3T3-L1 adipocytes expressing Myc-Rabip4 together with GFP-Rab4 (a-c), GFP-Rab5 (d-f), or GFP-Rab7 (g-i) or GFP-Rabip4 with myc-Rab11(j-l) were fixed and permeabilized. Panels a,d,g,j show the green labeling of the different GFP-fusion proteins. Panels b,e,h show labeling of myc Rabip4 and panel k shows myc Rab11. These myc-tagged protein are detected with anti-myc mAb followed by Texas-Red-coupled anti-mouse antibodies. Panels c,f,i,l are merged images. Bars, 1 µm.

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