Immunoblot analysis of arginase I and II, OAT, ODC, eNOS, and eNOS dimers and monomers

After culturing under hypoxic conditions for 48 h as described above, some PAECs were washed with PBS, covered with cell lysis buffer (Thermo Scientific, Rockford, IL, USA), protease inhibitor cocktail (Sigma-Aldrich), and phenylmethylsulfonyl fluoride (Sigma-Aldrich). The cells were scraped from the culture dish and centrifuged to separate the supernatant. Aliquots of the supernatant were stored at –80°C for later use in immunoblot analyses. Protein concentration for all homogenates was determined by Bradford assay.

For arginase I and II, OAT, ODC, and eNOS analysis, equal volumes of sample buffer composed of Tris-Glycine SDS 2× (Novex, Carlsbad, CA, USA) with 5% 2-mercaptoethanol (Sigma-Aldrich) were added to aliquots of the supernatant and heated to 80°C for 15 min. Using previously described methods, equal amounts of protein were loaded onto tris-glycine pre-cast 4–20% polyacrylamide gels (Invitrogen, Carlsbad, CA, USA) followed by electrophoresis at 120 volts for 2 h and then the protein was transferred to polyvinylidene difluoride membrane.¹⁵ To block non-specific protein binding, the membrane was then incubated at room temperature in PBS and 0.1% Tween-20 (PBS-T, Sigma-Aldrich) containing 5% non-fat dried milk and then washed in PBS-T containing 1% non-fat dried milk. To detect the protein of interest, the membrane was incubated overnight with arginase I (Abcam, Cambridge, MA, USA) 1:500, arginase II (Santa Cruz Biotechnology, Santa Cruz, CA, USA) 1:500, OAT (ThermoFisher, Waltham, MA, USA) 1:1000, ODC (Abcam) 1:500, or eNOS (BD Transduction Laboratories, San Jose, CA, USA) 1:1000, all diluted in PBS containing 0.1% Tween-20 and 1% non-fat dried milk as carrier buffer. After washing with PBS-T and 1% non-fat dried milk, the membrane was then incubated with anti-rabbit (for arginase I and II, OAT, and ODC; Cell Signaling Technology, Danvers, MA, USA) or anti-mouse (for eNOS; Cell Signaling Technology) IgG horseradish peroxidase (HRP)-linked secondary antibody diluted in the carrier buffer (1:2500) for 60 min. For eNOS dimers and monomers, we used nonsonicated and nonboiled lysates and low-temperature SDS-PAGE as previously described.¹⁶ Membranes were developed using enhanced chemiluminescence reagents (PerkinElmer, Waltham, MA, USA). The chemiluminescent signal was captured using an iBright FL1500 imaging system (ThermoFischer) and the bands for each protein were quantified using the iBright Analysis Software. After washing and stripping the membrane, we followed a similar procedure to re-probe the membranes for beta-actin (1:100,000) and anti-mouse IgG HRP-linked secondary antibody (1:2500, Cell Signaling Technology).