Fig. 6.

Stx6 functions during the re-entry of IRAP into the IRC following endocytosis from the cell surface. (A) Western blot showing the extent of Stx6 knockdown by siRNA (5 nmol) 72 hours after transfection. (B) Western blot showing the extent and specificity of Stx6 knockdown by siRNA (5 nmol) 96 hours after transfection. Lane 1, scrambled-siRNA-transfected cells; lane 2, Stx6-siRNA-transfected cells. For the 96-hour time point, the same membrane was stripped and re-probed with anti-Stx4, -Stx16 and -p115 antibodies. p115 is a Golgi-tethering factor and is used here as a protein loading control. (C) 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-hours recovery period, cells were stimulated with insulin and surface-labeled on ice with the anti-TfR monoclonal antibody. Following a 6-hour washout period 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 graph for clarity. a.u., arbitrary units.