Source was yeast cell line; bait produced as transgenic fusion protein; prey produced as transgenic fusion protein (prey from embryo cDNA expression library).

Two-hybrid system: yeast LexA-BD/LexA-AD

Source was yeast cell line; bait produced as transgenic fusion protein; prey produced as transgenic fusion protein (prey was previously cloned reagent).

Two-hybrid system: yeast LexA-BD/LexA-AD

Source was embryos of wild-type fly line; bait produced from endogenous gene; prey produced from endogenous gene.

Antibodies against Orc2 were used as controls and did not precipitate pnut.

Source was cell extract of L2 cell line; bait produced from transfected construct; prey produced from transfected construct.

Antibodies against Orc2 were used as controls and did not precipitate pnut.

Source was yeast cell line; bait produced as transgenic fusion protein; prey produced as transgenic fusion protein (prey was previously cloned reagent).

Two-hybrid system: "CytoTrap two-hybrid system provides an alternate strategy to detect protein-protein interactions in yeast. In this system, bait protein is fused with human son of sevenless (hSos) protein (Li et al., 1993), and a cDNA library or prey protein is expressed by fusion with myristoylation signal which anchors the prey fusion protein to yeast cell membrane. Protein interaction between bait and prey proteins recruits the hSos protein to the cell membrane, where hSos activates the Ras signaling pathway, leading to the survival of temperature-sensitive Saccharomyces cerevisiae (S. cerevisiae) strain cdc25H at 36 C. In the CytoTrap two-hybrid system, detection of protein interaction occurs in the cytoplasm near cell membrane and is not dependent on transcription activation of reporter genes. Hence, the system is particularly useful for identifying interaction partners of transcription factors and proteins that need post-translational modification in the cytoplasm, which could not be used as bait proteins in conventional transactivation-based yeast two-hybrid systems. Here we describe the construction of a cDNA library from the model plant Arabidopsis and a procedure for screening interaction proteins of AtSR1/CAMTA3, a Ca2+/CaM-regulated transcription factor from this library. This procedure could be adapted to identify interacting partners of interested proteins from other organisms."

Interaction in vitro; bait produced and labeled by in vitro translation; prey produced and labeled by in vitro translation.

Mutations to the GTPase domain of pnut (K155A, D206A, K288A) do not affect the interaction with Orc6.

Orc6 stimulates septin polymerization, binding filaments with a stoichiometry of 2.26 Orc6 molecules per filament repeat unit.

Source was cell extract of baculovirus-infected cell line; bait produced from transfected construct; prey produced from transfected construct.

Purified pnut-Sep1-Sep2 proteins were allowed to form filaments in the presence of purified Orc6. Binding of Orc6 to filaments was detected using Ni-NTA-Nanogold reagent and electron microscopy.