Figure 5. Protein-protein interaction of AtDMC1^G138D expressed using TMV-Gate vectors.

(A) Yeast two-hybrid analysis demonstrating self-interaction of AtDMC1^G138D. (B) Confirmation of AtDMC1^G138D self-interaction by pull-down experiment. The FLAG-tagged AtDMC1^G138D and 3xHA-tagged AtDMC1^G138D immobilized on anti-FLAG and anti-HA agarose beads, respectively, were incubated with total protein extract of N. benthamiana leaf discs transiently expressing c-myc-tagged AtDMC1^G138D or c-myc-tagged mGFP5 (negative control) and 3xFLAG-tagged AtDMC1^G138D or 3xFLAG-tagged GusPlus (negative control), respectively; corresponding TMV-Gate vectors and recombinant plasmids are listed in Table I. After washing the affinity matrices, proteins were eluted using SDS-PAGE sample buffer, resolved by SDS-PAGE and visualized by immunoblotting with antibodies as indicated. WB, western blot.