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. 2001 Mar 13;98(7):3837–3842. doi: 10.1073/pnas.061358098

Figure 5.

Figure 5

Effect of antisense menin on Smad heterodimerization, Smad nuclear accumulation, and formation of Smad/DNA complexes. (a) Effect of antisense menin (AS) on Smad2/3 and Smad4 heterodimerization. Myc-tagged Smad2 or Smad3 and flag-tagged Smad4 constructs were transfected (as indicated) into COS7 cells with or without antisense menin. Transfected cells were treated (+) or not (−) with 200 pM TGF-β for 1 h after overnight serum starvation. Cell extracts were immunoprecipitated with anti-Smad2/3 antibody followed by immunoblotting with anti-flag antibody. Expression of myc, flag, or menin was monitored. W, Western blot; IP, immunoprecipitation. (b) Effect of antisense menin (AS) on Smad2 and Smad3 nuclear accumulation. Nuclear extracts of Chinese hamster ovary cells transfected or not with the antisense menin were cultured in the absence or presence of 200 pM TGF-β and immunoblotted with Smad2/3, Stat3, and menin antibodies. V, empty vector. (c) Effect of antisense menin on the Smad3/4-DNA complex. GH4C1 cells were transfected with myc-Smad3 and flag-Smad4 in the presence or absence of antisense menin. Nuclear extracts were subjected to EMSA; a 29-bp double-stranded oligonucleotide derived from the PAI-1 promoter sequence of 3TP-Lux was used as probe. The shifted band is indicated by the arrow; it was decreased in intensity by antisense menin and by anti-myc antibodies (*). Menin and Smad3/4 in the nuclear extracts were monitored.