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. 2018 Jun 1;32(11-12):794–805. doi: 10.1101/gad.311936.118

Figure 1.

Figure 1.

HaloTagging the N termini of EZH2 and SUZ12 by genome editing: HaloTag-EZH2 maintains endogenous protein levels and intracellular PRC2 activity. (A, top) Diagram of the EZH2 or SUZ12 locus before and after 3xFlag-HaloTag genome editing. The 3xFlag-HaloTag is inserted downstream from the transcription start site (bent arrow) and in frame with the first codon of either EZH2 or SUZ12. The resulting gene editing generates an N-terminal 3xFlag-HaloTag fusion protein. PCR primers (horizontal arrows) bind outside of the left and right homology arms (LHA and RHA) and identify correctly edited alleles by a size increase of 1 kb, corresponding to the 3xFlag-HaloTag insertion. (Bottom) PCR of the EZH2 or SUZ12 locus in parental (P) or 3xFlag-HaloTag-edited cells. 3xFlag-HaloTag-EZH2 clone = dty3; 3xFlag-HaloTag-SUZ12 clone = 9.32. Parental genes give a 2-kb amplification, whereas the edited 3xFlag-HaloTag alleles give a 3-kb product. (B) Protein levels in parental and genome-edited cells were assessed by Western blots with αFlag, αEZH2, αSUZ12, and αHistone H3 antibodies. Based on molecular weight markers, the expected sizes of wild-type and 3xFlag-HaloTag fusion proteins are indicated at the right. (C) H3K27me3 levels in parental, 3xFlag-HaloTag-EZH2, and 3xFlag-HaloTag-SUZ12 cell lines. Cells were lysed in SDS lysis buffer, and αH3K27me3 and αH3 Western blots were performed on a dilution series (0.56×, 0.75×, and 1×) of the lysate.