Fig. 4

Demonstrating labeling specificity and efficiency in peroxisomes labeled with GCE-tagged GFP-SKL. HEK293T (a, b) or COS7 (c–f) cells were transfected with pBUD-Pyl-RS plasmids carrying the indicated GFP-SKL constructs and incubated for 48 h in the presence of the ncAA BCN-Lys (except for lanes 1 and 3 in b). Cells were then subjected to western blot analysis (a) or labeled with SiR-Tet for 1 h and subjected to in-gel fluorescence (b) or imaged live (c–g). b Cells were lysed, and equal total protein amounts were loaded on an SDS-PAGE. Results were recorded using SiR fluorescence. A specific band obtaining a similar size to that obtained in western blot was seen only in cells expressing GCE-tag-GFP-SKL and incubated with BCN-Lys, indicating specific labeling. c, d Maximum intensity projections of representative cells. Left to right: fluorescence obtained in each channel, merged image and co-localization analysis between intensity values of the 640 (SiR) and 488 (GFP) on a large subset of the cell that excludes the nucleus (white rectangle in merged image): Pearson’s correlation values: c 0.86, d 0.07. e Left to right: a zoomed-in image of the cell in c, a line intensity profile measured for the dashed line in the zoomed-in image, and a summary of SNR values measured for GFP and SiR in cells expressing the different versions of GFP-SKL. n = 40. f, g Cells expressing the indicated constructs were labeled with SiR-Tet and imaged using similar imaging parameters (exposure time and laser intensity). f GFP-positive peroxisomes were segmented based on intensity. For each peroxisome, the mean intensity in the GFP channel was plotted against the mean intensity in the SiR channel. ~ 95% of GFP-labeled peroxisomes exhibited SiR intensity levels that are above background. g SiR-positive puncta were segmented based on intensity. For each punctum, the mean intensity in the SiR channel was plotted against the mean intensity in the GFP channel. ~ 5% of SiR-positive puncta were GFP negative. Mean intensity levels measured for SiR puncta in cells expressing GCE-tag-GFP-SKL were at least twofolds higher than the levels measured for SiR puncta segmented in control cells expressing WT GFP-SKL, indicating specific labeling. Results were obtained in at least 3 independent experiments. Scale-bars 10 μm