Fig. 4: Mutations of the hinge residue Glu49 promoted uPAR dimerization on cell surface.

a Anti-uPAR immunoblotting of mRuby3-tagged uPAR mutants stably expressed in 293T cells. The cells were treated with the chemical cross linker BS3, washed, and lysed. Cell lysates were separated and analyzed by immunoblotting using a polyclonal anti-uPAR antibody (α-uPAR). Source data are provided as a Source Data file. b Microscopic images of uPAR-mRuby3 expressing 293 T cells treated with FITC-ATF (green) for 1 h and Hoechst (blue) for 10 min before fixation. Scale bars, 200 µm. c Quantification of ATF-binding ratio of b. ATF-binding ratio was measured by comparing the total intensity of FITC to the summed intensity of mRuby3 in each well. The summed intensity of FITC and mRuby3 were calculated by Operetta CLS software. Data were representative of three independent experiments. Data are presented as mean ± SD and the p-values of two-tailed unpaired Student’s t test are indicated. Source data are provided as a Source Data file. d uPAR dimerization changed uPAR membrane distribution. The uPAR distribution ratio was measured based on the mRuby3 intensity of apical layers (15 layers) and basal layers (5 layers) divided by the mRuby3 intensity of the whole cell (illustrated on the right) and analyzed by Operetta CLS software. These 20 layers covered 10 μm total vertical distance from the basal membrane. Source data are provided as a Source Data file.