Figure 8. Targeting mlCNBD-FRET to primary cilia.
(A) Strategy to target a protein to cilia. The somatostatin receptor 3 (SSTR3) has been fused to green fluorescent protein (eGFP) or mlCNBD-FRET. (B) Expression of eGFP in primary cilia of IMCD3 cells. An anti-acetylated tubulin antibody has been used as a marker for primary cilia. DNA has been labeled using DAPI. Scale bar is indicated. (C) Expression of mlCNBD-FRET in primary cilia of IMCD3 cells. Citrine fluorescence indicates the expression of mlCNBD-FRET. An anti-acetylated tubulin antibody has been used as a marker for primary cilia. DNA has been labeled using DAPI. Scale bar is indicated. (D) Representative image for HEK293 cells expressing SSTR3-mlCNBD-FRET. (E) Changes in FRET in HEK293 cells expressing SSTR3-mlCNBD-FRET (see D) after stimulation with 20 μM isoproterenol (black) or buffer only (grey). FRET has been measured using fluorescence microscopy. Data are presented as mean ± S.D.; n = 9 for each condition. (F) Acceptor photobleaching. The citrine (acceptor) fluorescence of mlCNBD-FRET in IMCD3 cells was bleached for 2 min with a mercury lamp using a 510/20 nm filter. A representative image is shown. The cerulean emission was recorded before and after acceptor photobleaching. Relative fluorescence intensities are color-coded from low (blue) to high (red). Scale bars are indicated.