Figure 4
Smad3 interacts with HIF-1α and bind to the VEGF promoter. (a) Immunoprecipitation of the Smad3/HIF-1α complex. Rat vascular SMCs were infected with either AdGFP or AdSmad3 followed by stimulation with TGF-β (5 ng/ml) for 6 h. Immunoprecipitation (IP) was then performed as described in the Materials and Methods; IgG was used for control. Immunoprecipitated complexes were detected by western blotting (WB) using antibodies against HIF-1α or phosphorylated Smad3. Shown is a representative blot from three independent experiments. (b) Proximal ligation assay showing interaction of HIF-1α and Smad3 in SMCs. Rat vascular SMCs were infected with either AdGFP or AdSmad3 followed by treatment with solvent or TGF-β (5 ng/ml) respectively for 6 h. The cells were then fixed and the interaction between Smad3 and HIF-1α was detected by Proximity Ligation Assay (red punctate marked by arrows) as described in the Materials and Methods. Shown are representative images each from three independent experiments; the lower panel shows enlarged nuclei (blue, DAPI stained). Scale bar is 5 μm. (c and d) Quantification of Smad3-HIF-1α interaction in SMCs. Proximal ligation was quantified as the number of ligation-positive punctate (red, see b) per cell (c) or per nucleus (d). (*P<0.05 compared with AdGFP control; n=3). (e) Rat vascular SMCs were infected with either AdSmad3 or AdGFP and then treated with TGF-β (5 ng/ml) for 6 h. Cells were crosslinked to fix the interaction between protein and DNA. The cell lysates were then sonicated to yield small DNA fragments between 200 and 700 bp. The samples were then incubated with Smad3, HIF-1α antibodies or control IgG. DNA bound to Smad3 or HIF-1α were recognized by antibodies and precipitated by protein A/G agarose beads. The precipitated DNA was measured by quantitative PCR using primer pairs to proximal or distal region of VEGF promoter. (*P<0.05 compared with IgG control; n=3)
