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Fig. 2 | Cell Communication and Signaling

Fig. 2

From: TEM8 functions as a receptor for uPA and mediates uPA-stimulated EGFR phosphorylation

Fig. 2

Identification of the region of uPA responsible for the interaction with TEM8. a Schematic representation of the full-length and truncated forms of uPA and full-length PA. EGFP was located at the C-terminus of the fusion proteins. b Representative laser scanning microscope FRET images. Paired constructs were co-transfected into HEK293 cells. Laser scanning microscope FRET images were captured from each of the three channels with the following excitation and emission wavelengths: EGFP (488/515 nm), FRET (488/590 nm) and RFP (543/590 nm). The labels on the top of each of the columns designate the three channels (green, FRET and red), then NFRET, and finally pixel-by-pixel corrected FRET (FRETc). The identity of the co-transfected constructs is indicated to the left of the panels. c The quantitative normalized FRET (NFRET) values were calculated from laser scanning microscope FRET images. The NFRET value is the average of regions of interest (ROIs) (n = 50–80) across three independent transfection experiments. ROIs were selected in areas that contained no protein aggregates. d Paired constructs were co-transfected into HEK293 cells. The cells were lysed, and protein extracts were subjected to SDS-PAGE and immunoblotting with anti-EGFP antibody (upper panel) or were immunoprecipitated with an anti-RFP antibody, and immunoblotted with an anti-EGFP antibody (lower panel). e & f Cells were cultured in 96-well cell culture plates and pre-treated with the several truncation mutants of uPA. The cells were fixed and treated with acidified buffer to remove endogenous uPA, then human HMW-tcuPA was added and the cell surface-based fibrinolytic activity was measured

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