Yeast Two-Hybrid Screening (y2h)

The Screening of TRβ against HeLa Library was performed in Saccharomyces cerevisiae strain L40 [trp-25, his3Δ200, leu2-3, ade2, LYS2::(lexAop) 4-HIS3, URA3::(lexAop)8lac GAL4], which contains the heterologous genes HIS3 and lacZ, according to a previous published study (35). The pBTMK-TRβ vector was used to express the full-length human TRβ, and pBTM116K-empty vector was used as a control. The autonomous activation of HIS3 gene was tested by co-transformation of yeast cells with pBTMK-TRβ and pACT2-empty vector (control), grown in minimal medium plates (without tryptophan, leucine, histidine) containing 5 mM 3-amino-1,2,4-triazole (3-AT). Also, we performed Beta-Galactosidase Test, in which colonies expressing Gal4 gene becomes blue, meaning that transcription factor was reconstituted by protein interaction. The y2h screenings were performed against HeLa cDNA library, cloned in pACT2 vector, expressing GAL4 activation domain (Matchmaker System, Clontech). After discovering the interaction partners, we performed co-transformation to confirm each interaction.