Dose response inhibition of ATB-BMPA photolabeling by cytochalasin B.

<p><i>A. ATB-BMPA photolabeling.</i> Ethanol (vehicle control) or cytochalasin B (CB) were added to 200 µg LDM for 10 min at room temperature. Samples were then irradiated with biotinylated ATB-BMPA (50 µM final concentration) as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025237#s2" target="_blank">Materials and Methods</a>”. Biotinylated proteins, isolated from detergent solubilized LDM using a high-capacity streptavidin agarose resin, were analyzed by immunoblot analysis using GLUT4 or GLUT1 antibodies. <i>B. Quantification of ATB-BMPA results.</i> GLUT proteins were quantified using an Odyssey Infrared Imaging System. Data, normalized to vehicle controls, represent the mean ± S.E. of three independent experiments. Half maximal inhibition (IC₅₀) of ATB-BMPA binding to GLUT4 and GLUT1 by CB were determined using a nonlinear least squares analysis (GraphPad Prism, v. 5.0).</p

Indinavir but not ritonavir or atazanavir selectively inhibits ATB-BMPA binding to GLUT4 relative to that of GLUT1.

<p>Indinavir, ritonavir, and atazanavir were added to 200 µg of LDM for 10 min at room temperature. ATB-BMPA (50 µM final concentration) was then added for 10 min at room temperature prior to UV irradiation. Biotinylated proteins were isolated as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025237#s2" target="_blank">Materials and Methods</a>” and then analyzed by immunoblot analysis using GLUT4 and GLUT1 antibodies. GLUT4 and GLUT1 protein were quantified using an Odyssey Infrared Imaging System. Data, normalized to vehicle treated controls, are shown as the mean ± S.E., n = 3; (*), p<0.05 for ATB-BMPA/GLUT4 vs. ATB-BMPA/GLUT1 binding as determined by the Student's t test.</p

Inhibition of ATB-BMPA photolabeling and glucose uptake by 10 µM HIV protease inhibitors.

<p><i>A, ATB-BMPA photolabeling</i>. Indinavir (Ind), atazanavir (Atz), ritonavir (Rit), and tipranavir (Tip) (10 µM final concentrations) were added to 200 µg of LDM for 10 min at room temperature. ATB-BMPA (50 µM final concentration) was then added for 10 min at room temperature prior to UV irradiation. Biotinylated proteins were isolated as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025237#s2" target="_blank">Materials and Methods</a>” and then analyzed by immunoblot analysis using GLUT4 and GLUT1 antibodies. <i>B, Quantification of ATB-BMPA results</i> GLUT4 and GLUT1 protein were quantified using an Odyssey Infrared Imaging System. Data, normalized to vehicle treated controls, are shown as the mean ± S.E., n = 3; (*), p<0.05 vs. vehicle as determined by the Student's t test; (+), p<0.05 for ATB-BMPA/GLUT4 vs. ATB-BMPA/GLUT1 binding. <i>C. Glucose Uptakes.</i> Indinavir (Ind), atazanavir (Atz), ritonavir (Rit), and tipranavir (Tip) (10 µM) were added 5–6 min prior to glucose uptakes in insulin-stimulated (1 µM insulin for 20 min) primary rat adipocytes and basal 3T3-L1 fibroblasts. [³H]2-Deoxyglucose uptake was measured at 37°C for 1 min in primary rat adipocytes (open bar) and for 6 min in basal 3T3-L1 fibroblasts (filled bar). Data shown as mean uptakes ± S.E. relative to control, n = 4; (*), p<0.05 vs. vehicle, (+), p<0.05 primary adipocytes vs. basal 3T3-L1 fibroblasts.</p

Half-maximal inhibition (IC₅₀) for ATB-BMPA binding to GLUT4 and GLUT1 by PI's.

<p>IC₅₀ values were determined from the dose response data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025237#pone-0025237-g005" target="_blank">Fig. 5</a>) using a nonlinear least squares analysis (GraphPad Prism, v. 5.0). (*), p<0.05 for IC₅₀ values for ATB-BMPA binding to GLUT4 vs. GLUT1 as determined by the Student's t test.</p

Inhibition of ATB-BMPA photolabeling and glucose uptake by 50 µM HIV protease inhibitors.

<p><i>A, ATB-BMPA photolabeling</i>. Indinavir (Ind), atazanavir (Atz), ritonavir (Rit), and tipranavir (Tip) (50 µM final concentrations) were added to 200 µg of LDM for 10 min at room temperature. ATB-BMPA (50 µM final concentration) was then added for 10 min at room temperature prior to UV irradiation. Biotinylated proteins were isolated as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025237#s2" target="_blank">Materials and Methods</a>” and then analyzed by immunoblot analysis using GLUT4 and GLUT1 antibodies. The effect of 20 µM cytochalasin B (CB) and no UV irradiation are shown for comparison. <i>B, Quantification of ATB-BMPA results</i> GLUT4 and GLUT1 protein were quantified using an Odyssey Infrared Imaging System. Data, normalized to vehicle treated controls, are shown as the mean ± S.E., n = 3; (*), p<0.05 vs. vehicle as determined by the Student's t test; (+), p<0.05 for ATB-BMPA/GLUT4 vs. ATB-BMPA/GLUT1 binding. <i>C. Glucose Uptakes.</i> Indinavir (Ind), atazanavir (Atz), ritonavir (Rit), and tipranavir (Tip) (50 µM) were added 5–6 min prior to glucose uptake in insulin-stimulated (1 µM insulin for 20 min) primary rat adipocytes and basal 3T3-L1 fibroblasts. [³H]2-Deoxyglucose uptake was measured at 37°C for 1 min in primary rat adipocytes (open bar) and for 6 min in basal 3T3-L1 fibroblasts (filled bar). Data shown as mean uptakes ± S.E. relative to control, n = 4; (*), p<0.05 vs. vehicle; (+), p<0.05 primary adipocytes vs. basal 3T3-L1 fibroblasts.</p

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

<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

Dose response inhibition of ATB-BMPA photolabeling by cytochalasin B.

<p><i>A. ATB-BMPA photolabeling.</i> Ethanol (vehicle control) or cytochalasin B (CB) were added to 200 µg LDM for 10 min at room temperature. Samples were then irradiated with biotinylated ATB-BMPA (50 µM final concentration) as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025237#s2" target="_blank">Materials and Methods</a>”. Biotinylated proteins, isolated from detergent solubilized LDM using a high-capacity streptavidin agarose resin, were analyzed by immunoblot analysis using GLUT4 or GLUT1 antibodies. <i>B. Quantification of ATB-BMPA results.</i> GLUT proteins were quantified using an Odyssey Infrared Imaging System. Data, normalized to vehicle controls, represent the mean ± S.E. of three independent experiments. Half maximal inhibition (IC₅₀) of ATB-BMPA binding to GLUT4 and GLUT1 by CB were determined using a nonlinear least squares analysis (GraphPad Prism, v. 5.0).</p