Fig. 3. uPAR promotes keratinocyte proliferation in vitro and is required for EGFR and ERK1/2 activation in cultured cells. (A,B) Primary keratinocytes from 2-day-old newborn wt and uPAR-KO littermates were grown in S-MEM containing 1% (A) or 8% (B) chelexed FCS, and their growth rate measured every day over a 7-day period. Values are expressed as absorbance at 650 nm after staining with 0.1% crystal violet in 200 mM MES, pH 6.0. The mean (± s.d.) of triplicate samples is reported. (C,D) Semi-confluent primary wt (C) and uPAR-KO (D) keratinocytes were grown in S-MEM 1% chelexed FCS in the presence or absence of EGF (10 ng/ml) and/or AG1478 (10 µM). The growth rate was measured every day over a 7-day period as described above. (E) The EGFR levels were evaluated by western blotting of wt and uPAR-KO cell lysates using an anti-EGFR antibody. (F) Semi-confluent primary wt and uPAR-KO keratinocytes were serum-starved for 18 hours and then stimulated with EGF (10 ng/ml) for 15 minutes. Cell lysates were immunoprecipitated with an antibody against EGFR and immunoblotted with anti-phospho-Tyr (pTyr), anti-EGFR and anti-uPAR antibodies. (G) Wt and uPAR-KO cell lysates were immunoblotted using antibodies against phospho-ERK1/2 (pERK1/2) and ERK2 (totERK). (H) Semi-confluent wt and uPAR-KO primary keratinocytes were serum-starved for 18 hours and stimulated with EGF (10 ng/ml) for 15 minutes. Cells were pre-treated for 20 minutes with AG1478 (10 µM). Protein extracts were then immunoblotted for phospho-ERK1/2 and ERK2 or immunoprecipitated with an anti-EGFR antibody and immunoblotted for phospho-Tyr, uPAR and EGFR.