Fig. 1
From: Identification of protein lysine methylation readers with a yeast three-hybrid approach
Schematic workflow of the Y3H experiment. The bait plasmid is composed of a GAL4-binding domain (GAL4-BD) fused with a histone 3 amino acids 1–20 (H3N) possessing the amino acid sequence that can be methylated at H3K9 by the HA-tagged catalytic domain of G9a enzyme (HA-G9a-SET). The prey plasmid is expressed as a fusion of GAL4-activation domain (GAL4-AD) and HA-tagged known reading domain of H3K9me3 (CBX1-CD, MPP8-CD) or human cDNA library. Haploid yeast strains transformed with pBRIDGE or pGADT7 expression plasmids were co-cultured to produce diploid yeast clones, expressing the bait and different prey proteins. The clone pool was plated on selective media to screen for interacting methylated bait with prey. Potential positive candidates were picked, patched, and grown further on selection media. Interacting preys were further isolated and the plasmids amplified in E. coli for sequencing of construct encoding region