FIGURE 2.

TUSC5 is a novel GSV protein. A, TUSC5 and GLUT4 expression was assessed in a panel of mouse tissues by Western blotting (representative image of n = 2). Asterisks denote TUSC5-specific bands of different molecular mass (* = ∼20 kDa, ** = ∼30 kDa). B, GSVs were immunoisolated from primary rat adipocytes treated with and without insulin. Abundance of GLUT4 and TUSC5 within the LDM fraction obtained from the pHS starting material and precipitates was determined by immunoblotting (representative of n = 3). C, GSVs were immunoisolated from 3T3-L1 adipocytes treated with and without insulin. GLUT4, IRAP, and TUSC5 levels were determined by immunoblotting (representative of n = 3). Two exposure levels are presented for TUSC5 to permit visualization of TUSC5 in the starting material. D, 3T3-L1 adipocytes were serum-starved for 2 h prior to fixation and processing for immunofluorescent imaging by confocal microscopy. Cells were stained for nuclei (DAPI, blue), TUSC5 (red), GLUT4 (green), and EEA1 (magenta). Cells were visualized at ×63 magnification. The basal surface (middle panel) and midpoint of specified region (cross-section, lower panel) are presented to aid visualization of colocalization. Instances of colocalization between GLUT4 and TUSC5 without EEA1 are indicated by closed arrowheads and colocalization between all three proteins is indicated by open arrowheads (top panel, scale bar, 50 μm, middle and bottom panel, scale bar, 10 μm). IP, immunoprecipitation; BAT, brown adipose tissue; Epi, epididymal fat.