Fig. 2: Real-time imaging of the EOF2 induced ectodomain shedding of Cherry-TGFα-GFP.

a Schematic representation of the mCherry-TGFα-GFP construct and mechanism of action. b Images of mCherry-TGFα-GFP expressing, serum-starved HEK293T cells stimulated with diluent, EOF2 (5 µM) or PMA (5 µM) during 180. mCherry/GFP ratio images at the indicates time points are shown in the intensity modulated display-mode. The colors range goes from red to blue to represent mCherry/GFP ratios. The upper and lower limits of the ratio range are shown on the right. Scale bar represent 20 µm. c Quantitative analysis of mCherry/GFP ratios in HEK293T cells expressing Cherry-TGFα-GFP. mCherry/GFP ratios were normalized to the average mCherry/GFP ratio measured before stimulation. The mean normalized mCherry/GFP ratios and SEM are shown, n = 40. See also supplementary movie 2. d Fluorescence analysis of m-Cherry in the culture medium of cells transfected with the fusion protein construct mCherry-TGFα-GFP as a ratio of GFP fluorescence in the cellular fraction. mCherry signal in the culture medium of transfected HEK293T cells was measured in a fluorimeter as described in the Material and Methods section. Bars represent the ratio red:green in transfected cells at different time points after treatment with EOF2 or EOF2 + PKC inhibitor Gö6850 as a percentage of the red:green ratio in non-treated transfected cells (dashed line). Cells were treated with the EOF2 at the indicated times. Data are the mean values ± S.E.M of nine independent measurements (n = 9). Statistical analysis: two tailed unpaired Student’s t test: (*PMA vs control at 60 min p = 0.0403; *PMA vs control at 180 min p = 0.0112).