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. 2003 Sep;23(17):6117–6128. doi: 10.1128/MCB.23.17.6117-6128.2003

FIG. 3.

FIG. 3.

Nuclear levels of USF1 and USF2 proteins decline with syncytiotrophoblast differentiation and are maintained at elevated levels by hypoxia and Mash-2 overexpression, while USF1 mRNA levels are unaffected. (A) Nuclear (N) and cytoplasmic (C) fractions were prepared from human trophoblasts before (Cyto) and after (Syn) culture for 3 days in 20% O2 or in an independent experiment after 3 days of culture in an atmosphere of 2 or 20% O2. Twenty micrograms of protein was analyzed for USF1 (43 kDa) and USF2 (44 kDa) by immunoblotting. (B) Nuclear (N) and cytoplasmic (C) fractions were prepared from human trophoblasts before culture (Cyto) or from trophoblasts that were infected with recombinant adenovirus expressing either Mash-2 (CMV-Mash-2; MOI = 5) or β-Gal (CMV-β-Gal; MOI = 5) and analyzed for USF1 and USF2 by immunoblotting (20 μg of protein/lane). (C) Total RNA isolated from human trophoblast cells cultured for 72 h in an atmosphere of 2 or 20% O2 or from trophoblast cells cultured in 20% O2 and infected with recombinant adenoviruses containing CMV-β-Gal or CMV-Mash-2 at an MOI of 0.5 to 5.0 was analyzed for hUSF1 mRNA levels by Northern blotting.