Hypoxia-induced Smad2 and Smad3 nuclear translocation in HUVECs.
HUVECs were grown to confluence on gelatin-coated chamber slides, serum starved overnight, and exposed for 1 hour to 1% O2 or 20% O2 with or without 100 pM recombinant TGF-β2. After cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100, immunostaining was performed with rabbit anti–Smad2 (10 μg/mL) followed by fluorescein isothiocyanate–conjugated donkey anti–rabbit IgG (3 μg/mL); or rabbit anti–Smad3 (10 μg/mL), followed by rhodamine-conjugated donkey anti–rabbit IgG (3 μg/mL). Immunofluorescence microscopy was performed with a Bio-Rad Radiance 2000 Confocal laser scanning microscope. Immunostaining for Smad2 is shown (A) in normoxic HUVECs, (B) after treatment with 100 pM TGF-β2, and (C) after exposure to hypoxia; immunostaining for Smad3 is shown (D) in normoxic HUVECs, (E) after treatment with 100 pM TGF-β2, and (F) after exposure to hypoxia. Original magnification, × 60.
