Hypoxia-induced Smad2 and Smad3 phosphorylation in HUVECs.
HUVECs were grown to confluence and were serum starved overnight, followed by exposure to 100 pM TGF-β2 for 30 minutes or to 1% O2 (hypoxia) for 10 minutes, 30 minutes, 1 hour, 2 hours, or 4 hours. As controls, untreated cells were simultaneously analyzed at the same time points (not shown). (A) Lysates were prepared from whole cells and quantified. Half were immunoprecipitated (IP)with goat anti–Smad2 antibody (top panel) and half with goat anti–Smad3 antibody (bottom panel) overnight at 4°C. Immunoprecipitates and molecular weight markers (not shown) were resolved by 10% SDS-polyacrylamide gels. Following electrophoresis and blotting, the membranes were developed by means of rabbit antibodies specific for the phosphorylated form of Smad2 (p-Smad2), total Smad2, phosphoserine (p-Smad3), or total Smad3 followed by chemiluminescence and autoradiography. Chemiluminescent bands were quantified using ImageQuant software. Shown are representative results from 1 of 3 independent experiments. (B) Hypoxia-induced fold increases in Smad phosphorylation or in total Smad protein were calculated by dividing the values for p-Smad2, p-Smad3, Smad2, and Smad3 found in hypoxic HUVECs by their respective normoxic control values. The graph presents mean fold changes (±SD) from 3 independent experiments (*P < .05).
