Nupage reducing reagent

Total protein from rat lung tissues was extracted using cell lysis buffer (Cell Signaling Technology, MA, USA) using the TissueLyser LT (Qiagen, Germany) for homogenization of the samples. Protein lysates from human PASMCs and PAECs were isolated using RIPA buffer (Thermo Fisher Scientific). Both buffers contained protease and phosphatase inhibitors (Calbiochem, Carlsbad, Germany). After centrifugation at 12,000g for 10 min at 4 °C, protein concentration was estimated and normalized using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). After denaturation for 10 min at 95 °C, equal amounts of protein (50 µg) were boiled in the presence of NuPAGE LDS Sample Buffer and NuPAGE Reducing Reagent and loaded on to 4–12% NuPage Bis-Tris Gels (Invitrogen, Carlsbad, CA, USA). Gel electrophoresis was followed by transfer to nitrocellulose membrane. Blocking was performed in 5% non-fat dry milk dissolved in 1% TBS/Tween-20 (TBS/T) at RT. Primary antibodies were incubated for overnight at 4 °C in 5% BSA in 1% TBS/T. All primary antibodies were diluted in 5% BSA diluted in Tris-buffered saline/Tween-20 for 1 h at room temperature and then incubated with primary antibodies at 4 °C overnight with gentle agitation. All primary antibodies used for western blot analyses are represented in Supplementary Table 1.

Cells were rinsed and pelleted, and the pellets were homogenized on ice in a cold Tris-EDTA+0.1% NP-40 lysis buffer. Lysates (15 μg) were reduced in NuPAGE reducing reagent (Invitrogen) and boiled for 5 min prior to loading in 4–12% NuPAGE Bis-Tris Gel for separation by electrophoresis. Proteins were transferred to nitrocellulose membranes and blocked with 3% bovine serum albumin (Thermo Scientific) in 1X TBST for 1 hour. The membrane was incubated with c-Myc overnight at 4°C followed by washing and incubation with horseradish peroxidase (HRP) secondary antibody. The protein bands were detected using ECL Plus (Amersham, GE Healthcare, Chicago, IL) on Amersham Imager 600 (GE Healthcare).