Fig. 1. uPA upregulates expression of C5aR in MCs. (A) RT-PCR analysis for C5aR mRNA was performed using the TaqMan method. RNA was isolated from quiescent MCs incubated for the indicated times with 20 nM uPA or in medium without uPA (control). GAPDH served as a house keeping gene. The results are presented as mean ± s.e.m. of four separate and independent experiments. (B) The quiescent MCs were stimulated for 20 hours at 37°C with the indicated concentrations of uPA, and C5aR protein was visualized in the membrane fractions by immunoblotting with anti-C5aR antibodies. MCs incubated in medium without uPA served as a control. The data on the upper panel are representative of four separate and independent experiments. Quantification of the results of these experiments by densitometry presented as mean ± s.e.m. is shown in the lower panel. Significance between control unstimulated and stimulated cells was determined by Student's t-test (*P<0.05; **P<0.01). (C) FACS analysis of MCs stimulated (solid line) or not (dashed line) for 20 hours at 37°C with 20 nM uPA using a FITS-labeled murine monoclonal anti-C5aR antibody. FITS-labeled isotype-matched antibody was used as a negative control (grey area). The results are representative of two separate and independent experiments. (D) The quiescent MCs were stimulated for 20 hours with 20 nM uPA or in medium without uPA (control). The cells were then fixed and stained with primary anti-C5aR antibodies and Alexa Fluor 488-conjugated secondary antibodies. Results are representative of three separate experiments.