Figure 6.

Effect of tyrosine phosphorylation of Src kinases on bleomycin (BLM)-induced ATII cell epithelial–mesenchymal transitions (EMT) in WT, urokinase-type plasminogen activator (uPA)-, uPA plasma membrane receptor (uPAR)-, and plasminogen activator inhibitor (PAI-1)–deficient mice. A–D: ATII cells isolated from WT (A), uPA-deficient (B), uPAR-deficient (C) and PAI-1–deficient (D) mice were treated with saline or BLM with or without caveolin-1 scaffolding domain peptide (CSP) or control peptide (CP) in culture dishes. The lysates were tested for changes in the phosphorylation of tyrosine (Y⁴¹⁸ and Y⁵²⁷) residues of Src kinases by Western blot analysis using phospho-specific antibodies. The same membrane was stripped and tested for total Src kinase and β-actin proteins. ATII cell lysates treated with TGF-β were also used as controls. E: ATII cells were treated with PBS, BLM, BLM in the presence of 10 μmol/L Src kinase inhibitor PP2 or control PP3 in culture dishes for 72 hours. The lysates were tested for changes in tyrosine Y⁴¹⁸ phosphorylation of Src kinases, PAI-1, uPA, collagen-I, α-SMA, E-cadherin, ZO-1, and β-actin by Western blot analysis. F: ATII cells isolated from WT mice were transduced with retrovirus expressing dominant-negative Y⁴¹⁸F mutant Src kinase. ATII cells from WT mice exposed to empty vector (EV) were used as controls for comparison. After 24 hours, these cells were treated with BLM, and the lysates were analyzed for changes in EMT markers 72 hours after BLM injury. WT ATII cells treated with PBS or BLM alone were used as controls. The lysates were analyzed for changes in EMT markers. G: ATII cells isolated from WT and uPA-deficient mice were exposed to saline or BLM or BLM with CSP or CP for 72 hours, and the lysates were immunoblotted for total and phosphorylated caveolin-1 (P-Cav-1), and β-actin using specific antibodies. H: ATII cells were treated with PBS, TGF-β, or TGF-β in the presence of 10 μmol/L inhibitor of TGF-β receptor kinase (SB-431542) in culture dishes for 72 hours. The lysates were immunoblotted for changes in tyrosine phosphorylation of Src kinases, PAI-1, uPA, collagen-I, α-SMA, E-cadherin, ZO-1, and β-actin by Western blot analysis.