Figure 6.

N-terminus p115 expression blocks insulin-stimulated GLUT4 and IRAP translocation. (A) Constructs consisting of 100 μg of pCDNA3.1 EGFP, N-terminal p115 EGFP, and C-terminal p115 EGFP were electroporated into 3T3-L1 adipocytes as described in Materials and Methods. After 48 h, cells were serum-starved for 4 h and stimulated without or with 100 nM insulin for 15 min. The cells were fixed with 3.7% formaldehyde and stained with antipolyclonal goat GLUT4 antibody followed by Cy3 antibody as described in Materials and Methods. (B) Electroporated cells (100 each from 3 cover slips) were scored for translocation and analyzed with Excel software. Statistical significance was determined by a Student's t test; ^*p > 0.01. (C) After insulin stimulation, the cells were stained antipolyclonal rabbit IRAP antibody followed by Cy3 antibody, and the data were analyzed (D) as in B. (E) Electroporation was performed as in A, and the cells were stained with the indicated antibodies followed by appropriate secondary antibodies labeled with Cy2 (GLUT4) and Cy5 (GM 130). The figure is representative of four independent experiments.