Figure 8.
GP changes in the local membrane environment surrounding the insulin receptor caused by insulin require the tyrosine kinase activity of the insulin receptor. (A) Tyrosine-phosphorylation of the KD mutant insulin receptor was inhibited upon insulin. Cells stably expressing the WT 2031-ACP-IR (left) or the KD mutant 2031-ACP-IR (right) were serum-starved for 2 h, then treated with 100 nM of insulin at 37°C for 5 min. Tyrosine-phosphorylation of ACP-IR was analyzed by Western blotting using α-insulin receptor β antibody (upper bands) and α-phospho-tyrosine antibody (lower bands). Molecular weights were indicated in the right side. (B) The differential delta GP suggested that the local membrane environment of the KD mutant insulin receptor showed no GP change by insulin treatment (red), whereas GP in the local membrane environment surrounding the WT insulin receptor dropped after insulin stimulation (blue). Stably expressing the WT 2031-ACP-IR or the KD 2031-ACP-IR cells were serum-starved for 2 h, followed by labeling with CoA-PEG5-NR, then spectral imaging was performed and the control solution or insulin solution were added at 120 s. The differential delta GP with insulin treatment of both WT and KD over the basal control-treated delta GP of each sample was plotted. The basal control-treated delta GP was estimated as the mean delta GP of 11 time-lapse data sets for both WT and KD with. Data represent means ± SEM of 13 time-lapse data sets for WT and 15 for KD. Significant change between WT and KD was statistically analyzed; during 0–120 s, ns, after the insulin treatment at 120 s, the change was significant *, P < 0.05. (C) The differential delta GP was analyzed in cells stably expressing WT 2031-ACP-IR in the presence of DMSO (control), 0.2 μM Latrunculin B, or 50 μM LY294002. The basal control-treated delta GP was calculated as the mean of 8 time-lapse data for control, 11 for Latrunculin B, and 9 for LY294002. Data present means ± of 8 time-lapse data for control, 11 for Latrunculin B, and 10 for LY294002. (D) AKt-phosphorylation upon insulin stimulation in the presence or absence of LY294002. 2031-ACP-IR-expressing cells were serum-starved for 2 h followed by incubation with serum-free medium, medium including 50 μM of LY294002 or DMSO for 10 min at 37°C, then cells were stimulated by 100 nM of insulin at 37°C for 5 min and phosphorylation of Akt was accessed by Western blotting analysis using α-Akt antibody (upper bands) and α phospho-Akt antibody (second upper bands). Actin was used as a loading control (lower bands). (E) Actin cytoskeleton was disrupted by Latrunculin B treatment. Cells plated on glass-bottom dishes were stained with Sir-actin at 37°C for 1 h, followed by washing excess Sir-actin, then cells were treated by DMSO (upper) or 0.2 μM of Latrunculin B (lower) at 37°C for 10 min. Left pictures are Nomarski images and right pictures are images of Sir-actin. Scale bar, 10 μm.

GP changes in the local membrane environment surrounding the insulin receptor caused by insulin require the tyrosine kinase activity of the insulin receptor. (A) Tyrosine-phosphorylation of the KD mutant insulin receptor was inhibited upon insulin. Cells stably expressing the WT 2031-ACP-IR (left) or the KD mutant 2031-ACP-IR (right) were serum-starved for 2 h, then treated with 100 nM of insulin at 37°C for 5 min. Tyrosine-phosphorylation of ACP-IR was analyzed by Western blotting using α-insulin receptor β antibody (upper bands) and α-phospho-tyrosine antibody (lower bands). Molecular weights were indicated in the right side. (B) The differential delta GP suggested that the local membrane environment of the KD mutant insulin receptor showed no GP change by insulin treatment (red), whereas GP in the local membrane environment surrounding the WT insulin receptor dropped after insulin stimulation (blue). Stably expressing the WT 2031-ACP-IR or the KD 2031-ACP-IR cells were serum-starved for 2 h, followed by labeling with CoA-PEG5-NR, then spectral imaging was performed and the control solution or insulin solution were added at 120 s. The differential delta GP with insulin treatment of both WT and KD over the basal control-treated delta GP of each sample was plotted. The basal control-treated delta GP was estimated as the mean delta GP of 11 time-lapse data sets for both WT and KD with. Data represent means ± SEM of 13 time-lapse data sets for WT and 15 for KD. Significant change between WT and KD was statistically analyzed; during 0–120 s, ns, after the insulin treatment at 120 s, the change was significant *, P < 0.05. (C) The differential delta GP was analyzed in cells stably expressing WT 2031-ACP-IR in the presence of DMSO (control), 0.2 μM Latrunculin B, or 50 μM LY294002. The basal control-treated delta GP was calculated as the mean of 8 time-lapse data for control, 11 for Latrunculin B, and 9 for LY294002. Data present means ± of 8 time-lapse data for control, 11 for Latrunculin B, and 10 for LY294002. (D) AKt-phosphorylation upon insulin stimulation in the presence or absence of LY294002. 2031-ACP-IR-expressing cells were serum-starved for 2 h followed by incubation with serum-free medium, medium including 50 μM of LY294002 or DMSO for 10 min at 37°C, then cells were stimulated by 100 nM of insulin at 37°C for 5 min and phosphorylation of Akt was accessed by Western blotting analysis using α-Akt antibody (upper bands) and α phospho-Akt antibody (second upper bands). Actin was used as a loading control (lower bands). (E) Actin cytoskeleton was disrupted by Latrunculin B treatment. Cells plated on glass-bottom dishes were stained with Sir-actin at 37°C for 1 h, followed by washing excess Sir-actin, then cells were treated by DMSO (upper) or 0.2 μM of Latrunculin B (lower) at 37°C for 10 min. Left pictures are Nomarski images and right pictures are images of Sir-actin. Scale bar, 10 μm.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal