SMAD5β does not heterodimerize or homodimerize.

The protein interactions of SMADs were assayed by the yeast 2-hybrid system. Various SMAD forms were expressed in frame with the LexA DNA binding domain in the pHyb-LexA plasmid (bait): the full-length forms, SMAD4, SMAD5, and SMAD5β; the polymorphic form SMAD5β.N345S; and a truncated form, SMAD5.e6t. Each SMAD was also expressed in frame with the transcription activation domain B42 in the pYEST-B42AD plasmid (prey). Transformants were tested for protein interactions using the β-galactosidase filter assay. The intensity of β-galactosidase was scored based on the intensity of the blue color, ranging from no color (−) to strongly positive (+++).