Figure 1.

Experimental setup and gating strategy of FRET measurements. Clover, mRuby2, Clover + mRuby2 and Clover fused (63bp) mRuby2 protein expression are shown in total HEK293T cell lysates by Western blot analysis against GFP (A) and tRFP (B). Fluorescence microscopy images (C) of Clover, mRuby2, Clover + mRuby2 and Clover fused (63bp) mRuby2 expressing HEK293T cells. Panel 1 (1) shows the brightfield. Cells expressing mRuby2 are depicted in panel 2 (2), Clover-positive cells in panel 3 (3). Cell nuclei were counterstained with Hoechst 33342 (panel 4 (4)). An overlay to estimate cytosolic and nuclear region is provided in panel 5 (5). FLIM images (D) of HEK293T cells with the controls Clover, mRuby2, Clover + mRuby2 and Clover fused (63bp) mRuby2. The histogram (E) shows the normalized frequency of fluorescence lifetimes in the images. Experimental setup and gating strategy to measure FRET by flow cytometry are depicted in living cells (F). HEK293T cells were stably transduced with the controls Clover, mRuby2, Clover + mRuby2 as well as the Clover fused (63bp) mRuby2 and analyzed using a flow cytometer. Double positive cells were gated in panel 1 (1). False-positive FRET signals resulting from mRuby2 excitation by the 488 nm laser were excluded in panel 2 (2). In panel 3 (3), the remaining cells were evaluated for FRET by adjusting a gate defining cells which were co-transduced with Clover + mRuby2 and should be FRET-negative. The numbers in panel 3 (3) give total percentages of FRET-positive HEK293T cells. Images and flow cytometry-plots are representative for experiments which were performed at least three times. (G) FRET efficiencies were determined as described in Materials and Methods by analyzing Clover/mRuby2-double positive cells only. Experiments were performed at least three times. ***P ≤ 0.001.