Fig. 7.
Interaction of TRB4-5 with various partners. (A) The Y2H system was used to assess protein–protein interactions of TRB4-5 proteins with TERT fragments, RUVBLs and POT1a/b as in Fig. 6. Co-transformation with an empty vector (AD, BD) served as a negative control. Asterisks, 1 mM 3-aminotriazol (3-AT); cross, 3 mM 3-AT. (B) Interactions between N-terminal domain of PWO1-3 and TRB4-5 were detected as in A). Interactions with full length PWO1-2 proteins are in Supplementary Fig. 8B. (C) Novel interactions between TRB4-5 and EMF2/VRN2, as well as interactions with SWNΔSET/CLFΔSET were tested using Y2H system as in A). Novel interactions were verified by Co-IP. The TNT expressed VRN2 and EMF2 (35S-labelled*) were mixed with TRB4-5 (myc-tag) and incubated with anti-myc antibody. In the control experiment, the VRN2 and EMF2 proteins were incubated with anti-myc antibody and Protein G magnetic particles in the absence of partner protein. Input (I), unbound (U), and bound (B) fractions were collected and separated in SDS–10% PAGE gels