Figure 2.

Mutation of a conserved arginine in the MH1 domain does not interfere with Smad2/Smad4 heteromeric interaction. COS-1 cells were transfected with combinations of empty vector (−), Flag-tagged wild-type (wt) or mutant (mut) Smad2 (1:2 DNA ratio) or Smad4 (1:1 DNA ratio in A; 1:4 DNA ratio in B), and wild-type (wt) or activated (act) TGF-β type I receptor (TβRI). Cell lysates were subjected to immunoprecipitation with α-Flag antibody and Smad2-associated Smad4 was visualized by α-HA immunoblotting (α-Flag IP, α-HA blot). Total protein levels were determined by immunoblotting of total cell lysates (α-Flag or α-HA blot).