Antibody against α sma

PASMCs were identified by expression of α-actin (≥ 95% of the cells) by immunofluorescence using an antibody against α-SMA (1:200, Abcam, USA). Primary human pulmonary artery SMCs (ScienCell Research Laboratories, USA) were maintained in a Petri dish containing prechilled PBS, 2% penicillin–streptomycin, Ca²⁺-free HBSS, and 0.5% fetal calf serum mixed with 20% DMEM/F12 medium (Billups-Rothenberg, Del Mar, USA). Then, the cells were cultured in an atmosphere containing 5% CO₂ and 21% O₂ or 3% O₂ in a humidified incubator at 37 °C for 3, 6, 12, 24, and 48 h for normoxia or hypoxia treatments, respectively. For cell proliferation, Ca²⁺ imaging, and detection of the phosphorylation signaling pathway, PASMCs were pretreated with cyclopiazonic acid (CPA), nifedipine (Nifed), EGTA (Santa Cruz, USA), BAPTA/AM (Santa Cruz, USA), LY294002 (Santa Cruz, USA), Perifosine (Santa Cruz, USA), LY317615 (Santa Cruz, USA), rapamycin (Santa Cruz, USA), or PD98059 (Santa Cruz, USA) and seeded onto 25 mm glass coverslips as indicated. Then, various concentrations of recombinant human RELM-β protein (0–40 ng/ml, Abgent, USA) were used to stimulate PASMCs for various periods (0–72 h). Cells of passages 3–7 were used in the experiments. When cells in the logarithmic growth phase reached a density of 70%, cell cycle was synchronized by incubation in the serum-free medium for 24 h.

For the immunofluorescence studies, the lungs were collected, frozen, and embedded in OCT (Jung, Japan). Eight-micrometer tissue sections were obtained, and the α-SMA protein was assessed using an antibody against α-SMA (Abcam, UK) and revealed with a secondary antibody anti-mouse FITC (Acris, Germany). The α-SMA was observed in green color. The nuclei were stained with DAPI. Tiling fluorescent microscopy was used to assess more than 10% of each section and 2000 cells counted for each condition.