Figure 4

Expression of fluorescent fusion proteins. A. Schematic representation of the fusion proteins produced in order to test the feasibility of our toolkit, indicating the micrograph panels in Figure 4B where each protein’s expression is shown. The relative positions of the nuclear location signals (N1, N2) and nuclear export signals (E1, E2) in murine wild type (wt) SIRT1 is shown. ΔN-tSIRT1, N-terminal-deleted murine SIRT1 (see main text); the portion deleted from the wt isoform is indicated. Fusion proteins are clustered in functional sets, as described in the main text. B. Detection of fluorescent protein fusions under an epifluorescence microscope. Panels a-d: expression of PAR-2 fused to mKate2 (a,b) or EGFP (c,d) in transiently-transfected HeLa cells under control conditions (a,c) or after treatment with the PAR2-specific agonist AC55541 (5 μM, 1 h; panels b, d). White bar: 10 μm. Panels e, f: Differential localisation of wt- (e) or ΔN-tSIRT1 (f) proteins fused to mKate2 in transiently-transfected HeLa cells, under control conditions. White bar: 10 μm. Panels (g-j): expression of two p65/RelA-based fusions with mKate2 after transient trasfection of their expression vectors into Raw264.7 monocytes, under control (g,i) or LPS-stimulated conditions (100 ng/ml, 1 h; panels h,j). White Bar: 10 μm. All micrographs were taken at 200X magnification. C . Frames from a time-lapse movie of a Raw264.7 monocyte transfected with the expression vector for the PAR2-mKate2 fusion protein and imaged on a confocal microscope (63X objective lens) after adding 5 μM AC55541 in order to monitor cellular trafficking of the receptor. The complete time-lapse experiment is provided as Additional file 5: Movie S1. Time elapsed after adding the agonist is indicated in each frame. The white arrow in the 20 min-panel points at some PAR2-mKate2-labelled intracellular vesicles that are seen trafficking in Additional file 5: Movie S1.