spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

Right arrow Help viewing high resolution images
Right arrow Return to article
(Downloading may take up to 30 seconds.
If the slide opens in your browser, select File -> Save As to save it.)
Click on image to view larger version.


Figure 1


Fig. 1. Newly synthesized IRAP-TfR-AA76,77 defaults to the plasma membrane, where it accumulates under steady-state conditions. (A) Differentiated 3T3L1 adipocytes were electroporated with 50 µg of IRAP-TfR-WT (panels a and c) or IRAP-TfR-AA76,77 (panels b and d) and, 16 hours later, cells were serum-starved and treated without (panels a and b) or with (panels c and d) 100 nM insulin for 20 minutes. The cells were then fixed, permeabilized and labeled with an anti-TfR antibody followed by Texas-Red-conjugated secondary antibody, and were then visualized with a Zeiss LSM510 scanning laser confocal microscope. (B) The data in A were quantified as the ratio of surface fluorescence:total fluorescence, as described in the Materials and Methods. White bars, basal cells; black bars, insulin-stimulated cells. (C) Differentiated 3T3L1 adipocytes were electroporated with 50 µg of IRAP-TfR-AA76,77 and, 16 hours later, cells were incubated with vehicle alone (panels a-d) or vehicle plus 10 µg/ml cycloheximide (CHX) (panels e-h) for 0, 1, 2 or 4 hours. The cells were then processed for confocal microscopy as indicated in A. (D) The data presented in C were quantified by calculating the ratio of plasma membrane:total fluorescence, as described under Materials and Methods. Results are from three independent experiments, with fifteen cells per experiment (mean ± s.e.m.). (E) Fully differentiated 3T3L1 adipocytes were transfected with 50 µg IRAP-TfR-WT or 50 µg IRAP-TfR-AA76,77 cDNA. After an overnight recovery period, cells were incubated for 0, 0.5, 1, 2, 4 or 6 hours in 10 µg/ml CHX. In lane 1, cells were plated immediately in CHX after electroporation and were then incubated for 6 hours. At the indicated time points, cell lysates were prepared and subjected to western blotting. An anti-IRAP antibody was used to detect both endogenous IRAP (upper panel) and the expressed IRAP-TfR chimera (lower panel). a.u., arbitrary units.





Right arrow Return to article