Fig. 5
From: One pattern analysis (OPA) for the quantitative determination of protein interactions in plant cells
FLIM analysis of AP1-mV NLS-mCh and AP1-mV Ap1-mCh in N. benthamiana leaf cells. FLIM experiments were performed in N. benthamiana leaf epidermis cells. Fusion proteins were expressed from the UBQ10 promoter and imaged 3–4 days after infiltration. A Average lifetime image of individual nuclei expressing AP1-mV, AP1-mV NLS-mCh and Ap1-mV AP1-mCh (A’–A’’ respectively). Nucleoli were excluded from FLIM analysis. (Scale bars: A–A’’: 6 µm) B BINDING [%] (grey) and FRET efficiencies [%] (white) for AP1-mV, AP1-mV NLS-mCh and AP1-mV AP1-mCh. For each analysed nucleus average BINDING and a corresponding average lifetime were fitted. Mean BINDING of the donor only sample AP1-mV was 1.23% and most values were below 10%. Therefore, nuclei with BINDING below 10% were excluded from FRET efficiency calculation. Co-expression of NLS-mCh with AP1-mV did not lead to significant higher BINDING (3.06%) compared to the donor only sample, while AP1-mV AP1-mCh showed increased BINDING (29.89%) with an average FRET efficiency of 39.98%. Statistical groups were assigned after multiple comparison with Kruskal–Wallis and a Post hoc test using the criterium Fisher’s least significant difference (alpha parameter is 0.05). (Dashed blue line marks the BINDING cut-off of 10%; Number of repetitions are indicated below BINDING values and number of images with BINDING above 10% are indicated below the FRET efficiency values in the bottom of the plot)