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Fig. 7. Stx6 function is not required for the insulin-stimulated exit of IRAP from the IRC. (A) Fully differentiated 3T3L1 adipocytes were transfected with 5 nmol of scrambled or Stx6 siRNA and, 72 hours later, were transfected with 50 µg EGFP-IRAP-TfR-WT reporter. Following a 24-hour recovery period, cells were treated with or without insulin, fixed and labeled with Texas Red secondary antibody. The ratio of plasma membrane to total fluorescence was determined using Zeiss LSM software. (B) Cells were transfected with either 5 nmol of scrambled or Stx6 siRNA and, 72 hours later, were transfected with IRAP-TfR-WT reporter. Following an 18-hour recovery period, cells were stimulated with insulin and surface-labeled on ice with anti-TfR monoclonal antibody. Following a 6-hour washout at 37°C, cells were re-stimulated with or without insulin, fixed, labeled with Texas Red secondary antibody and processed for confocal microscopy (mean ± s.e.m. of three independent experiments). A timeline for the transfections is shown above the bar graphs for clarity. Scram, scrambled siRNA. a.u., arbitrary units.
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