Fig 8.

Smad3 and PSmad1/5 bind BREs in vitro in response to costimulation with TGF-β and BMP7. (A and B) Nuclear extracts were prepared from HaCaT cells that had been starved overnight in Opti-MEM and then treated for 1 h with the ligands shown. They were analyzed either directly by Western blotting using antibodies against Smad2/3, PSmad1/5, PSmad3, and Smad4 as a control (Inputs) or by DNAP using an oligonucleotide containing mutated Smad binding sites (32) (mutant), one corresponding to a single copy of the BRE sequences that drive the BRE-luciferase reporter (BRE) and one corresponding to the −1070/−1025 region of the ID1 promoter (A) or the mutant oligonucleotide, oligonucleotides corresponding to the −2979/−2920 (probe A) and −2919/−2860 (probe B) regions of the ID2 promoter (B).