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1.
FIGURE 1.

FIGURE 1. From: Sac3 Is an Insulin-regulated Phosphatidylinositol 3,5-Bisphosphate Phosphatase.

Sac3 is up-regulated in 3T3L1 adipocytes versus 3T3L1 fibroblasts. RIPA++ lysates were collected on the indicated day of the 3T3L1 cell differentiation program. Equal protein amounts (100 μg) along with an HA-Sac3 standard were analyzed in duplicate by immunoblotting (IB) with the indicated antibodies. A decrease in tubulin levels (∼50%) is typical during differentiation. Shown is a representative experiment.

Ognian C. Ikonomov, et al. J Biol Chem. 2009 September 4;284(36):23961-23971.
2.
FIGURE 4.

FIGURE 4. From: Sac3 Is an Insulin-regulated Phosphatidylinositol 3,5-Bisphosphate Phosphatase.

Sac3 depletion enhances insulin-induced glucose transport. 3T3L1 adipocytes were transfected with the indicated siRNA duplexes. After 64 h, serum-starved cells were stimulated with insulin at the indicated concentrations followed by the 2DG uptake assay. Data expressed relative to the basal control indicate significant increases in 2DG uptake under Sac3 silencing; means ± S.E. from 3 independent experiments in triplicate; *, p < 0.01.

Ognian C. Ikonomov, et al. J Biol Chem. 2009 September 4;284(36):23961-23971.
3.
FIGURE 6.

FIGURE 6. From: Sac3 Is an Insulin-regulated Phosphatidylinositol 3,5-Bisphosphate Phosphatase.

Sac3 phosphatase activity is inhibited by insulin. Serum-starved 3T3L1 adipocytes were treated with or without insulin (100 nm; 10 min) and lysed in RIPA++ buffer. Anti-Sac3 immunoprecipitates (500 μg of protein) were analyzed for phosphatase activity with the indicated di-C8-PIs substrates by a malachite green assay. Reactions were carried out in triplicate and are presented as the released inorganic phosphate; means ± S.E. of 3–4 independent experiments for each substrate; *, p < 0.01 versus unstimulated control.

Ognian C. Ikonomov, et al. J Biol Chem. 2009 September 4;284(36):23961-23971.
4.
FIGURE 7.

FIGURE 7. From: Sac3 Is an Insulin-regulated Phosphatidylinositol 3,5-Bisphosphate Phosphatase.

Sac3 is phosphorylated in vivo in an insulin-independent manner. Serum-starved 3T3L1 adipocytes were labeled with [32P]orthophosphate, treated with or without insulin (100 nm; 10 min), and lysed in RIPA++ buffer, supplemented with 1 mm pervanadate and 50 nm okadaic acid. Aliquots were immunoprecipitated (IP) with preimmune (P), anti-PIKfyve, or anti-Sac3 sera as indicated. Immunoprecipitates were washed, resolved by SDS-PAGE, and electrotransferred onto nitrocellulose membranes that were subjected to autoradiography. The identity of the proteins is confirmed by subsequent immunoblotting with anti-PIKfyve, anti-Sac3, or anti-ArPIKfyve antibodies (mobility indicated by arrows). Insulin has no effect on Sac3, PIKfyve, and ArPIKfyve phosphorylation in two to three independent cell labelings. The slight increases in the phosphorylated bands under insulin (+) are due to slightly greater protein amounts (∼20%) subjected to immunoprecipitation versus control (−).

Ognian C. Ikonomov, et al. J Biol Chem. 2009 September 4;284(36):23961-23971.
5.
FIGURE 8.

FIGURE 8. From: Sac3 Is an Insulin-regulated Phosphatidylinositol 3,5-Bisphosphate Phosphatase.

HPLC analysis of PIs in radiolabeled cells. A, inositol- and serum-starved 3T3L1 adipocytes labeled with myo-[3H]inositol (40 h) were treated with or without insulin (100 nm; 10 min). Lipids were extracted, deacylated, and fractionated by HPLC in the presence of [32P]GroPIns standards. Presented is the radioactivity from the phosphatidylinositol 4,5-bisphosphate region of a representative experiment. Insulin increased [3H]inositol-PtdIns(3,5)P2 by 21.4 ± 1.8%; n = 3; p < 0.05. B, 3T3L1 adipocytes were transfected with the indicated siRNAs. Three days after transfection cells were serum-starved and labeled with [32P]orthophosphate (2.5 h). Deacylated lipids were analyzed by HPLC with an on-line flow scintillation analyzer. 32P radioactivity integrated from the counts within the elution times corresponding to the [32P]GroPIns peaks determined by the 3H-labeled standards were summed (total PI radioactivity). [32P]PtdIns(3,5)P2 and [32P]PtdIns(3)P were calculated as a percentage of total PI radioactivity; means ± S.E., n = 4; *, p < 0.05.

Ognian C. Ikonomov, et al. J Biol Chem. 2009 September 4;284(36):23961-23971.
6.
FIGURE 2.

FIGURE 2. From: Sac3 Is an Insulin-regulated Phosphatidylinositol 3,5-Bisphosphate Phosphatase.

Sac3 depletion enhances insulin-induced GLUT4 cell-surface accumulation. A, 3T3L1 adipocytes transfected with indicated siRNA duplexes were lysed 60 h post-transfection. Duplicate samples immunoblotted (IB) with the indicated antibodies show ∼60% reduction in Sac3 protein but not in PIKfyve or ArPIKfyve. B and C, 3T3L1 adipocytes were cotransfected with the indicated siRNAs and HA-GLUT4-eGFP cDNA. After 60 h, serum-starved cells were treated with or without insulin (1 nm; 20 min), stained with anti-HA and Cy3-conjugated anti-rabbit IgG without permeabilization, and analyzed by confocal microscopy. B, typical images depicting GFP fluorescence and exofacial HA (Cy3) staining in basal (panels a, b, e, and f) or insulin-stimulated cells (panels c, d, g, and h) under control (panels a, b, c, and d) or Sac3 depletion (panels e, f, g, and h). C, cell-surface HA (Cy3) signal-to-total GFP fluorescence ratio in HA-GLUT4-eGFP-expressing cells by quantitative fluorescence microscopy (means ± S.E.; 4 independent experiments; *, p < 0.01 versus insulin-stimulated control group).

Ognian C. Ikonomov, et al. J Biol Chem. 2009 September 4;284(36):23961-23971.
7.
FIGURE 5.

FIGURE 5. From: Sac3 Is an Insulin-regulated Phosphatidylinositol 3,5-Bisphosphate Phosphatase.

Unaltered Sac3-PIKfyve and Sac3-ArPIKfyve assembly by insulin. Serum-starved 3T3L1 adipocytes were treated with or without insulin (100 nm) for the indicated time intervals (A) or for 10 min (B). Aliquots were immunoprecipitated (IP), resolved by SDS-PAGE, and immunoblotted (IB) as indicated. Shown are chemiluminescence detections of immunoblots from representative experiments out of 3–7 independent experiments with similar results. A, aliquots (800 μg of protein) of the RIPA++ buffer-lysed cells were immunoprecipitated with preimmune (Preim) or anti-PIKfyve. B, cells were fractionated to isolate IM, PM, and cytosol. Equal percentage (92 ± 1.9%) of each RIPA detergent solubilized fraction (protein content: IM, 93 μg; PM, 60 μg; cytosol, 512 μg) was immunoprecipitated with anti-PIKfyve antibodies. C, cells were fractionated to isolate IM, PM, and cytosol. Equal percentage (90 ± 1.7%) of the RIPA detergent solubilized fractions (protein content: IM, 106 μg; PM, 63 μg; cytosol, 772 μg) was immunoprecipitated with a preimmune serum (P) or anti-ArPIKfyve antibodies (I). Electrophoretic mobility of Sac3 and ArPIKfyve standards (St.) is denoted. A and B, membranes were cut horizontally and immunoblotted. C, membrane was stripped between the antibodies.

Ognian C. Ikonomov, et al. J Biol Chem. 2009 September 4;284(36):23961-23971.
8.
FIGURE 3.

FIGURE 3. From: Sac3 Is an Insulin-regulated Phosphatidylinositol 3,5-Bisphosphate Phosphatase.

Expression of Sac3WT, but not phosphatase-deficient Sac3D488A, inhibits GLUT4 surface translocation by insulin. 3T3L1 adipocytes were cotransfected with Myc7-GLUT4 and the indicated eGFP-Sac3 cDNA constructs. After 24 h, serum-starved cells were treated with or without insulin (100 nm, 20 min) and stained with anti-Myc followed by Alexa568-conjugated anti-mouse IgG without permeabilization. Cells were then permeabilized, incubated with the C-terminal GLUT4 antibodies followed by Alexa350-conjugated anti-rabbit IgG, and analyzed by confocal microscopy. A, typical confocal images of insulin-stimulated cells depicting expressed eGFP-Sac3 (panels a and d), exofacial Myc (panels b and e), and total Myc7-GLUT4 (panels c and f). B, ratio of cell-surface Myc (Alexa568) to total Myc7-GLUT4 fluorescence (Alexa350) in Myc7-GLUT4-expressing cells by quantitative fluorescence microscopy (means ± S.E.; 3 independent experiments; *, p < 0.01 versus insulin-stimulated control). C, quantitation of cell-surface Myc fluorescence by counting cells displaying a complete plasma membrane rim and presented as a percentage of Myc7-GLUT4 positive cells (∼100 cells/condition) in 3 independent experiments; means ± S.E.; *, p < 0.01 versus the other two insulin-stimulated groups.

Ognian C. Ikonomov, et al. J Biol Chem. 2009 September 4;284(36):23961-23971.

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