Figure 1.

HEC Proteins Interact with PIFs.

(A) Quantitative yeast two-hybrid assay shows the interactions among the HEC1/HEC2 and members of the PIF family. LacZ assays were performed in triplicate and the data represent mean ± se. β-Galactosidase units are Miller units (M.U.). GAD, GAL4 activation domain; GBD, GAL4 DNA binding domain.

(B) HEC2 heterodimerizes with PIF1 and PIF3 in vitro. The gel photograph shows the input and the pellet fractions. Full-length HEC2 ORF fused to GAD was used for this coimmunoprecipitation assay as described (Huq and Quail, 2002; Toledo-Ortiz et al., 2003; Shen et al., 2005). All proteins were synthesized as ³⁵S-Met-labeled products in TnT reactions.

(C) PIF1 interacts with HEC2 in in vivo coimmunoprecipitation assays. The input and pellet fractions are indicated. Total protein was extracted from 4-d-old dark-grown seedlings. Coimmunoprecipitations were performed using the anti-GFP antibody, and the immunoprecipitated samples were probed with both anti-myc and anti-GFP antibodies.

(D) HEC2 preferentially interacts with the unphosphorylated form of PIF1 in in vivo coimmunoprecipitation assays. The input and pellet fractions are indicated. Total protein was extracted from 4-d-old dark-grown seedlings or dark-grown seedlings exposed R light. Coimmunoprecipitations were performed using the anti-GFP antibody, and the immunoprecipitated samples were probed with both anti-myc and anti-GFP antibodies.