Protein Extraction and Western Blot Analysis

Preparation of membrane and cytosolic proteins from lung tissue and HPASMCs, separation by SDS-PAGE under reducing conditions, and transfer of proteins to nitrocellulose membranes were performed as described previously [4 (link)]. After staining the membranes with Ponceau S (Sigma) and blocking the nonspecific binding sites, blots were exposed to primary antibodies: monoclonal mouse anti-nestin (Chemicon; 1:1000), mouse anti-nestin (BD Transduction; 1:1000), mouse anti-nestin (Santa Cruz; 1:200), mouse anti-vinculin (Sigma; 1:30000), mouse anti-β-actin (Sigma; 1:20000), mouse anti-SMA (Sigma; 1:5000), polyclonal rabbit anti-PDGFR-β (Upstate, Schwalbach, Germany; 1:500), rabbit anti-phospho-PDGFR-β (p-PDGFR-β) (Santa Cruz; 1:250), rabbit anti-poly (ADP-ribose) polymerase 1 (PARP1) and rabbit anti-cleaved PARP (Cell Signaling Technology, Frankfurt, Germany; 1:1000). After washing, the membranes were probed with peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Pierce, Rockford, IL, USA; 1:2000). Immunoreactive bands were visualised by enhanced chemiluminescence [4 (link)]. Densitometric quantification was performed using ImageJ software (NIH, Bethesda, MD, USA).