Analysis of FANCA/FANCG binding interaction by yeast 2-hybrid method.

(A) Diagrams of the constructs used in the yeast 2-hybrid assays. FANCA 1-230 was subcloned in-frame, downstream of the GAL4 activation domain (AD) (amino acids 768-881) in the pACT2 vector. FANCGwt and FANCG (amino acids 134-278) were subcloned in-frame, downstream of the GAL4 DNA-binding domain (GAL4BD) (amino acids 1-147) in the pAS2-1 vector. (B) Colony-lift filter assay to assess the binding between FANCA and FANCG proteins. Yeast strain Y190 was cotransformed with the plasmids indicated. The expression of proteins was confirmed by immunoblotting of whole yeast extract with antibodies against GAL4-AD or GAL4 DNA-BD (data not shown).