Western Blot Analysis

HumanPASMC were cultured in a 6-cm dish at a density of 6000 cells/ cm2 and cultured for 48 hours in serum-free DME. The cells were washed twice with PBS, and then placed in DME supplemented with 10% FBS and antibiotics. The cells were then cultured in normal or hypoxic oxygen concentrations for the indicated times with or without NO donors or BPS. After incubation, the cells were harvested and resuspended in protein lysis buffer (150mM of NaCl, 20mM of Tris-HCl, 1% NP40, 10mM of EDTA, 10% glycerol, 1mM of PMSF, 10 |g/ml of aprotinin, 1 |g/ml of leupeptin, 1 |g/ml of gestating) and then incubated for 30 minutes on ice. After incubation, the cell lysis buffers were centrifuged at 10,000g for 15 minutes at 4°C to remove the cell fragments and the supernatants were analyzed for protein content using a DC protein assay kit. Each sample was quantified and then 25 | g of protein was loaded onto each lane of a 4 - 12% Bis-Tris Nupage gel with MES SDS running buffer, according to the manufacturer's protocol. The gel was transferred to a PVDF membrane by electrophoresis at 100 V for 1 hour. The membrane was blocked in PBS, 0.2% Tween 20 (PBS-T), and 5% non-fat milk at room temperature for 1 hour. All antibodies were diluted in the same blocking buffer. The membrane was then probed either with rabbit anti-p21, anti-p27 or anti-p53 polyclonal antibody (1:1000 dilution) or mouse anti-s-actin monoclonal antibody (1:5000 dilution) and then incubated for 1 hour at room temperature. After incubation, the membrane was washed with PBS-T and incubated with horseradish peroxidase-conjugated goat anti-rabbit or mouse IgG (1 : 2000 dilution) for 2 hours at room temperature. After washing with PBS-T, an ECL system was used for detection of the proteins. Each assay was performed in 4 independent experiments.

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