Labeling glucose transporters with biotinylated ATB-BMPA using low-density microsomes.

Abstract

<p>LDM were isolated from fully differentiated 3T3-L1 adipocytes as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025237#s2" target="_blank">Materials and Methods</a>”. In (<i>A</i>), 175 µg of intact or Thesit-solubilized LDM were immunoprecipitated overnight with 25 µg of control rabbit IgG or 25 µg of GLUT4 C-terminal antibodies precoupled to Protein A-agarose. The supernatant (sup) and pellets were analyzed by immunoblot analysis using a monoclonal GLUT4 antibody (Cell Signaling). In (<i>B</i>), ∼1% ethanol (vehicle control) or 20 µM cytochalasin B (CB) were added to the indicated amount of LDM for 10 min at room temperature. Samples were then incubated for an additional 10 min in the dark at room temperature with biotinylated ATB-BMPA (250 µM final concentration). UV irradiation was then performed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025237#s2" target="_blank">Materials and Methods</a>”. Excess photolabel was removed using a spin desalting column. Membranes were solubilized in 2% Thesit detergent buffer and biotinylated proteins were isolated using a high capacity streptavidin agarose resin, and immunoblotted with anti-GLUT4 antibody. GLUT4 protein was quantified using an Odyssey Infrared Imaging System. Membranes containing biotinylated ATB-BMPA that were not irradiated with UV are shown for comparison. In (<i>C</i>), biotinylated ATB-BMPA (50 µM final concentration) was added to 70 µg of LDM for varying times prior to UV irradiation. Biotinylated proteins were isolated as described in <i>A</i>, and then analyzed by immunoblot analysis using GLUT4 and GLUT1 antibodies. Glut proteins were quantified using an Odyssey Infrared Imaging System.</p