Figure 1.
AtNF-YB-AtNF-YC interactions by colony yeast two hybrids assays.
A.The indicated AtNF-YCs were fused to the Activation Domain (AD) and tested with AtNF-YB fused to the DNA-binding domain of GAL4. B. Same as A, except that the reverse experiment was tested, namely the AtNF-YBs fused to the Activation Domain were matched to the AtNF-YCs fused to the DNA Binding Domain. ++ refers to robust growth on the selective medium, + weak growth, and − no growth.
Figure 2.
AtNF-YB-AtNF-YC interactions by liquid yeast two hybrids assays.
Yeast two hybrids assays in liquid cultures using the AtNF-YB (DBD) and AtNF-YC (AD) configuration are depicted. β-Galactosidase Units were measured as detailed in Materials and Methods. The experiments were repeated three times and the standard deviations are indicated.
Figure 3.
AtNF-Y Subunits interactions in vitro.
A. The indicated labelled, TnT produced NF-YCs were assayed in affinity assays with recombinant AtNF-YB2 containing an His-tag. Load (L), flow-.through (FT) and bound (B) fractions of NTA Nickel columns, with (Lanes 2 and 3) and without (Lanes 4 and 5) His-AtNF-YB2 were run on SDS-PAGE gels and labelled proteins were revealed by autoradiography. B. Same as A, except that labelled, TnT produced AtNF-YA6 was added to the load fraction.
Figure 4.
EMSAs of AtNF-Y subunits with mouse NF-Y.
A.Electrophoretic Mobility Shift Assay of the indicated AtNF-YB with recombinant mouse NF-YA and NF-YC using a labeled CCAAT-containing oligonucleotide. B. Same as A, except that At NF-YCs were used with recombinant mouse NF-YA and NF-YB. C. Same as A, except that AtNF-YA were used with recombinant mouse NF-YB and NF-YC. The migration of the mouse NF-Y complex is indicated.
Figure 5.
Electrophoretic Mobility Shift Assay of the indicated AtNF-Y subunits with a labelled CCAAT-containing oligonucleotide.
Figure 6.
E. coli co-expression of LEC1/AtNF-YB9 with AtNF-YC3 allows functional heterodimerization, heterotrimerization and CCAAT-binding.
A. Purification of soluble LEC1/AtNF-YB9 or L1L/AtNF-YB6 HFD heterodimers by co-expression with AtNF-YC3. Nickel-affinity purification elution profiles obtained from soluble fractions of 6His-LEC1/AtNF-YB9 or 6His-L1L/AtNF-YB6 with AtNF-YC3. Equal volumes of indicated elution fractions (E) in 100 mM Imidazole of LEC1/AtNF-YB9 or L1L/AtNF-YB6 with AtNF-YC3 were analysed by SDS-PAGE and Coomassie staining. E2, were dialysed and used in Agarose gel non-radioactive EMSAs shown in (B). B. Fluorescence agarose gel EMSAs of trimer reconstitution with mouse NF-YA. 5′-Cy5 labeled CCAAT oligonucleotide probe was incubated with increasing amounts of the indicated 6His-tagged HFD dimers isolated by Ni-affinity purification, or mouse 6His-NF-YB/NF-YC as positive control, in the presence, or absence, of purified mouse NF-YA. Purified (untagged) mouse NF-Y trimer was used as a reference for NF-Y complex migration.