Ha-Ras-ERK 1/2-ROS signaling in fibroblasts from patients with cGVHD. (A) Levels of reactive oxygen species, evaluated as DCF fluorescence intensity (arbitrary units) in 3 fibroblast cell lines from patients with and without GVHD (ecGVHD and No GVHD, respectively). Fibroblasts from patients with ecGVHD also were pre-incubated with selective inhibitors of EGFR and PDGFR (AG 1478 and AG 1296, respectively; 2 μM for 2 hours). (B) Left part. The data shown were obtained with 2 and 4 fibroblast cell lines from patients with and without GVHD (ecGVHC and No GVHD, respectively) cultured in 0.2% FCS for 48 hours before being harvested. Immunoblots were developed with specific antibodies against Ha-Ras on cell lysates immunoprecipitated with a monoclonal anti–pan-Ras antibody. Phosphorylated forms of ERK 1/2 were detected by immunoblotting with phosphospecific antibodies. The right part of the panel shows the densitometric analysis of the blots of the left part.