Figure 1.
Strategy for monitoring the local membrane environment surrounding the insulin receptor and validation of membrane localization of the dye. (A) Schematic image of monitoring the local membrane environment surrounding the insulin receptor using ACP-tag and CoA-derivatized Nile Red. The probe fluorescence is extremely low in medium and when covalently attached to the ACP-tag on the receptor, then Nile Red becomes strongly fluorescent (orange). (B) Structure of CoA-PEG-NR with various PEG-lengths, n = 5, 11, 14, and 27. (C) Schematic of constructs used. Domain structures of insulin receptor α chain and the cartoon of whole insulin receptor depicting ACP-tag positions. (D) Specific labeling of ACP-insulin receptor in the plasma membrane with CoA-PEG11-NR. Cyan is DAPI staining in nucleus and magenta is CoA-PEG11-NR staining. Scale bar, 10 μm. (E) Emission spectral shift depending on distance from ACP-tag on the receptor to the plasma membrane. Cells expressing each ACP-IR were stained with CoA-PEG11-NR or NR12S and emission spectrum between 561 and 695 nm was measured from five images for 1992-ACP-IR (n = 59 ROIs), 2031-ACP-IR (n = 37), and PreCT-ACP-IR (n = 69), and from seven images for NR12S (n = 42). Normalized fluorescent intensities were plotted. (F) Sensitivity of emission spectrum to cellular-cholesterol modification. Cell expressing each ACP-IR were treated with 10 mM of cyclodextrin for 15 min or 2.5 mM of cholesterol-cyclodextrin for 1 h at 37°C followed by labeling with CoA-PEG11-NR or NR12S, then the emission spectrum was measured. Data represents the mean ± SEM from 5 images (n = 57–59 ROIs for 1992-ACP-IR, n = 36–37 for 2031-ACP-IR and n = 69–75 for PreCT-ACP-IR), and the mean ± SE from 28 images of NR12S (n = 168 ROIs for each sample).

Strategy for monitoring the local membrane environment surrounding the insulin receptor and validation of membrane localization of the dye. (A) Schematic image of monitoring the local membrane environment surrounding the insulin receptor using ACP-tag and CoA-derivatized Nile Red. The probe fluorescence is extremely low in medium and when covalently attached to the ACP-tag on the receptor, then Nile Red becomes strongly fluorescent (orange). (B) Structure of CoA-PEG-NR with various PEG-lengths, n = 5, 11, 14, and 27. (C) Schematic of constructs used. Domain structures of insulin receptor α chain and the cartoon of whole insulin receptor depicting ACP-tag positions. (D) Specific labeling of ACP-insulin receptor in the plasma membrane with CoA-PEG11-NR. Cyan is DAPI staining in nucleus and magenta is CoA-PEG11-NR staining. Scale bar, 10 μm. (E) Emission spectral shift depending on distance from ACP-tag on the receptor to the plasma membrane. Cells expressing each ACP-IR were stained with CoA-PEG11-NR or NR12S and emission spectrum between 561 and 695 nm was measured from five images for 1992-ACP-IR (n = 59 ROIs), 2031-ACP-IR (n = 37), and PreCT-ACP-IR (n = 69), and from seven images for NR12S (n = 42). Normalized fluorescent intensities were plotted. (F) Sensitivity of emission spectrum to cellular-cholesterol modification. Cell expressing each ACP-IR were treated with 10 mM of cyclodextrin for 15 min or 2.5 mM of cholesterol-cyclodextrin for 1 h at 37°C followed by labeling with CoA-PEG11-NR or NR12S, then the emission spectrum was measured. Data represents the mean ± SEM from 5 images (n = 57–59 ROIs for 1992-ACP-IR, n = 36–37 for 2031-ACP-IR and n = 69–75 for PreCT-ACP-IR), and the mean ± SE from 28 images of NR12S (n = 168 ROIs for each sample).

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal