Figure 4.
CoA-PEG5-NR linked ACP-insulin receptor detects changes in the local membrane environment surrounding the insulin receptor caused by insulin. (A) Tyrosine-phosphorylation of 2031-ACP-IR upon insulin in the presence or absence of attachment of PEG11-NR. 2031-ACP-IR expressing cells were serum-starved for 2 h followed by incubation with serum-free medium, medium including SFP synthase, or medium including SFP synthase and CoA-PEG11-NR for 30 min at room temperatures, then cells were stimulated by 100 nM of insulin at 37°C for 5 min and phosphorylation of 2031-ACP-IR was accessed by Western blotting analysis using α-insulin receptor β antibody (left upper bands) and α-phospho-tyrosine antibody (left middle bands). Molecular weights were indicated in the right side. Tyrosine-phosphorylation levels are presented as the mean fold increase over the basal ±SEM, n = 7–8, for each condition. Statistical significance was calculated by one-way ANOVA, ns, not significant. (B) Time course of mean delta GP change compared to time 0. After 2 h serum starvation, cell stably expressing 2031-ACP-IR were labeled with CoA-PEG5-, 11-, or 27-NR or NR12S and cells transiently expressing ACP-GPI were stained with CoA-PEG11-NR. Spectral imaging was performed every 10 s and the control solution and insulin solution were added at 60 and 240 s, respectively. GP value was calculated with two emission regions, 561–597 and 605–641 nm, and the delta GP compared to the time 0 at each time point was plotted. Data represents the mean ± SEM from 19 time-lapse image sets for CoA-PEG5-NR labeled ACP-IR, 22 for CoA-PEG11-NR-labeled ACP-IR, 17 for CoA-PEG27-NR-labeled ACP-IR, 12 for labeled with NR12S, and 19 for CoA-PEG11-NR-labeled ACP-GPI. Source data are available for this figure: SourceData F4.

CoA-PEG5-NR linked ACP-insulin receptor detects changes in the local membrane environment surrounding the insulin receptor caused by insulin. (A) Tyrosine-phosphorylation of 2031-ACP-IR upon insulin in the presence or absence of attachment of PEG11-NR. 2031-ACP-IR expressing cells were serum-starved for 2 h followed by incubation with serum-free medium, medium including SFP synthase, or medium including SFP synthase and CoA-PEG11-NR for 30 min at room temperatures, then cells were stimulated by 100 nM of insulin at 37°C for 5 min and phosphorylation of 2031-ACP-IR was accessed by Western blotting analysis using α-insulin receptor β antibody (left upper bands) and α-phospho-tyrosine antibody (left middle bands). Molecular weights were indicated in the right side. Tyrosine-phosphorylation levels are presented as the mean fold increase over the basal ±SEM, n = 7–8, for each condition. Statistical significance was calculated by one-way ANOVA, ns, not significant. (B) Time course of mean delta GP change compared to time 0. After 2 h serum starvation, cell stably expressing 2031-ACP-IR were labeled with CoA-PEG5-, 11-, or 27-NR or NR12S and cells transiently expressing ACP-GPI were stained with CoA-PEG11-NR. Spectral imaging was performed every 10 s and the control solution and insulin solution were added at 60 and 240 s, respectively. GP value was calculated with two emission regions, 561–597 and 605–641 nm, and the delta GP compared to the time 0 at each time point was plotted. Data represents the mean ± SEM from 19 time-lapse image sets for CoA-PEG5-NR labeled ACP-IR, 22 for CoA-PEG11-NR-labeled ACP-IR, 17 for CoA-PEG27-NR-labeled ACP-IR, 12 for labeled with NR12S, and 19 for CoA-PEG11-NR-labeled ACP-GPI. Source data are available for this figure: SourceData F4.

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