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Fig. 6. uPA signaling requires uPAR–β1-integrin interactions. (A) Time course of ERK activation in H1299 and H1264 cells. Cells were serum-starved for 24 hours, acid-washed to remove bound endogenous uPA and then incubated with pro-uPA (10 nM) in serum-free medium for different time points (minutes): 0, 0.5, 1, 5, 10, 30. The cells were lysed in RIPA buffer and immunoblotted with anti-phospho-ERK and anti-total-ERK antibodies, respectively. (B) uPA-induced signaling in H1299 cells. H1299 cells expressing WT or HD uPAR were serum-starved, acid washed and then incubated with pro-uPA (10 nM) in serum-free medium for 5 minutes to assess ERK activation and 30 minutes to assess activation of Akt, GSK-3β or Stat. The lysates were immunoblotted with anti-phospho- and anti-total-antibodies to ERK, Akt, GSK-3β, or Stat1 or Stat3. (C) Effects of inhibitors on uPA-induced signaling. H1299 and H1264 cells expressing endogenous uPAR were serum-starved, acid washed, pretreated with uPAR–β1-integrin blocking peptide (β1P1) or scrambled peptide (scβ1P1) and then incubated with pro-uPA (10 nM) in serum-free medium. (D) EGF-induced ERK phosphorylation is not affected by H249A and/or D262A mutations in uPAR. H1299 cells expressing WT, H, D or HD uPAR were serum-starved and incubated with 20 ng/ml EGF for 10 minutes and cell lysates were assessed for ERK activation. All the experiments were performed at least three times with similar results.
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