Fig 2. Image analyses of heterodimerization between TASK1 and TALK2.
(A) A BiFC assay was performed to detect direct coupling between TASK1 and TALK2. A representative set of images from three independent experiments are presented. HEK293 cells expressing VN-TALK2+TALK2-VC or VN-TASK5+TASK1-VC were used as positive and negative controls, respectively. (B) FRET analysis based on acceptor photobleaching was carried out using a confocal microscope. YFP within the ROI 1–5 (white rectangles in the figures) in HEK293 cells co-expressing TASK1-YFP+CFP-TALK2 was selectively photobleached using a laser at 514 nm wavelength. (C) Fluorescence intensity of YFP and CFP in the ROIs was obtained and compared before and after YFP photobleaching. Note that the increase in CFP fluorescence, i.e. the cancellation of FRET, was detected together with the decrease in YFP fluorescence. EFRET values (calculated based on an equation mentioned in Materials and Methods) of ROI 1–5 are 7.4, 4.8, 6.6, 6.0 and 10.1%, respectively. (D) EFRET values were compared between cells co-expressing TALK2-YFP+CFP-TALK2, TASK1-YFP+CFP-TASK1, TASK1-YFP+CFP-TALK2, and TASK5-YFP+CFP-TASK1. Cells were transfected with cDNA at a ratio for CFP:YFP of 1:1. Numbers of cells are shown in parentheses. **p<0.01 vs. CFP-TASK1+TASK5-YFP. (E) Co-IP assay was performed using HEK293 cells expressing GFP-TASLK2 + TASK1 (without any tag) or only TASK1 (for a negative control). Lysates were precipitated with anti-GFP antibody, and blotted using the anti-TASK1 antibody. Each protocol was repeated three times and showed very similar results.