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Fig. 3. Overexpression of Rip11 inhibits insulin-stimulated GLUT4 translocation to, and fusion with, the plasma membrane. (A) 3T3-L1 adipocytes were electroporated with plasmids encoding HA-GLUT4-GFP in the presence of either mRFP-Rip11 or mRFP vector, as indicated. 24 hours later, the cells were treated in the absence or presence of 100 nM insulin for 30 minutes and were then fixed and stained with an antibody against HA to detect surface-localised GLUT4. The intensity of HA antibody staining was divided by the intensity of total cellular GFP fluorescence. This provides a ratio that represents the degree of fusion of GLUT4 with the plasma membrane, corrected for the expression level of GLUT4. (B) Representative images are provided of insulin-stimulated cells from the experiment in panel A (images of additional cells can be found in supplementary material Fig. S1). The cells were analysed for the effect of Rip11 overexpression (panels b and e; where panels a and d are from cells expressing an mRFP vector control) on insulin-stimulated GLUT4 translocation, as determined by the distribution of GFP (panels a-c, where panel c is expressed as the amount of GFP fluorescence in the plasma membrane divided by total cellular GFP fluorescence) and the extent of fusion of translocated GLUT4 with the plasma membrane (panels d-f; where panel f is expressed as the intensity of HA fluorescence in the plasma membrane divided by the intensity of GFP fluorescence found at the plasma membrane) in the presence of insulin. Both panels of the figure are representative of three separate preparations of adipocytes, with the data expressed as the mean ± s.e.m.
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