Pgadt7 ad

All SUMO CDS were translationally fused with the activation domain of pGADT7 AD (Takara Bio). βC1 was fused with binding domain and cloned into pGBKT7 BD (Takara Bio). Plasmids were transformed into AH109 strain as described previously (Gietz and Woods, 2002) Successful transformants were screened on -Leu, -Trp media followed by screening for interaction on -Leu,-Trp, -His with or without 3AT (Sigma-Aldrich). Successful interactions were further screened on -Leu, -Trp, -His, -Ade media.

Yeast transformation was performed as described with minor modifications (Gietz and Woods, 2002) . Freshly streaked AH109 cells were used to initiate primary culture grown overnight in YPD media (yeast extract 1%, bacterial peptone 2%, and dextrose 2%). Cells were grown to A600 = 0.6 OD. About 10 ml of cells were pelleted per transformation. The freshly pelleted cells were transferred to a 1.5 ml centrifuge tube and washed with deionized sterile water followed by 0.1 M lithium acetate. Transformation mixture (PEG 3000 50%, salmon sperm DNA and lithium acetate) was added to the washed cells, and cells were resuspended. The corresponding mixture of AD and BD plasmids was added to the transformation mixture followed by vortexing for 30 s. The mixture was incubated for 30 min at 30°C and 30 min at 42°C. The reaction mixture was removed and cells were resuspended in 2 ml YPD media and allowed to recover for 2h before plating onto an auxotrophic media. All Rec and Rad proteins were translationally fused with the Activation Domain (AD) of pGADT7 AD (Takara Bio). βC1 was fused with Binding Domain (BD) and cloned into pGBKT7 BD (Takara Bio). Plasmids were transformed into AH109 strain as described previously (Gietz and Woods, 2002) . Transformants were screened on -Leu, -Trp media followed by screening for interaction on -Leu,-Trp, -His with or without 3-AT (Sigma-Aldrich).