Figure 1.

14-3-3 λ Interacts with PHOT2 in Yeast and in in Vitro Pull-Down Assays.

(A) Yeast colonies expressing different combinations of the DNA binding domain (DB-) and activation domain (AD-) of the yeast GAL4 two-hybrid system. Only yeast cells coexpressing the PHOT2 and 14-3-3 λ constructs (2 and 3) activated the LacZ reporter gene (blue color). 1, DB-PHOT2 + AD-; 2, original 14-3-3 λ-interacting clone; 3, cotransformation of the DB-PHOT2 and the rescued AD-14-3-3 λ; 4, DB- + AD-14-3-3 λ.

(B) Arabidopsis14-3-3 λ interacts with PHOT2 in in vitro pull-down assays. Equal OD₂₈₀ readings of the affinity-purified bacterial MBP- and MBP-PHOT2 fusion protein were used to interact with [³⁵S]Met-labeled 14-3-3 λ (Promega TNT coupling system). Proteins were resolved in SDS-PAGE and blotted. The immunoblot (right panel) was reacted with anti-MBP antibodies. The signals of the retained [³⁵S]Met-labeled 14-3-3 λ by MBP- and MBP-PHOT2 were visualized by autoradiography (left panel). MBP-PHOT2 retained more [³⁵S]Met-labeled 14-3-3 λ, as it had a stronger signal than the MBP fraction alone.