P erk1

H&E staining, immunohistochemistry, and alcian blue staining were performed from paraffin-embedded tissue samples; 1–2 µm slides were cut by microtome (Leica). All antibodies for immunohistochemical staining were used in 1:50, 1:100, or 1:200 dilution, and incubated overnight at 4 °C. Primary antibodies: DNA polymerase theta (Abcam ab111218, Cambridge, UK), PARP1 (Abcam ab32138, Cambridge, UK), Mre11 (Cell Signaling 4895, Danvers, MA, USA), Ku70 (Santa Cruz Biotechnology sc-1486, Heidelberg, Germany), Ki67 (Bethyl Laboratories IHC-00375, Montgomery, TX, USA), PCNA (Cell Signaling 2586, Danvers, MA, USA), CyclinD1 (Cell Signaling 2978, Danvers, MA, USA), p-ERK1 (Cell Signaling 4370, Danvers, MA, USA), Cox2 (Cell Signaling 12282, Danvers, MA, USA). Alcian blue was performed by Alcian Blue pH 2.5 Stain Kit (Vector Laboratories H-3501, Newark, CA, USA), according to the manufacturer’s protocol. All slides used for histological analysis were scanned with the Pannoramic Midi II (3DHISTECH, Budapest, Hungary), and evaluated with different magnification using Quant Center software (3DHISTECH, Budapest, Hungary).

For the western blot analysis rat PASMCs were isolated from rat pulmonary arteries and seeded in 3.5 cm dishes until they reached a nearly confluent growth state. PASMCs were starved for 24 hours (0.1% BSA) and afterwards stimulated with PDGF-BB (10 ng/ml) in the absence or presence of NS1619 (100 µmol/l; 120 minutes preincubation, Sigma-Aldrich, Germany). After four and eight minutes, respectively, the cells were frozen in liquid nitrogen and subsequently scraped in triton-X-100 lysis buffer. Protein concentration was determined by Bradford assay. Extracted protein samples were boiled in a Laemmli buffer and 50 µg of each sample was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE; 10%) and transferred onto nitrocellulose membrane as described previously [8] (link). Proteins were detected using primary antibodies against p-ERK1, p-ERK2, ERK1, ERK2 and p-AKT (Cell Signaling, Danvers, USA;1∶1.000 each antibody) and appropriate secondary antibodies labeled with infrared dyes and visualized using the Odyssey infrared imaging system (Li-COR Biosciences, Bad Homburg, Germany). Densitometry was carried out using the integrated Odyssey software.